Cyclic peptide tyrosine-tyrosine conjugate coupled with glucagon-like peptide 1 (GLP-1) fusion peptide and application of cyclic peptide tyrosine-tyrosine conjugate

Abstract

The invention relates to a cyclic peptide tyrosine-tyrosine conjugate coupled with a glucagon-like peptide 1 (GLP-1) fusion peptide and application of the cyclic peptide tyrosine-tyrosine conjugate. The present invention includes a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide. The invention also relates to pharmaceutical compositions and methods of use thereof. The novel conjugates are useful for preventing, treating, or ameliorating diseases and disorders disclosed herein.

Description

[0001] This application is a divisional application of an invention application with an application date of April 24, 2019, a Chinese national application number of 201980028055.4, and an invention name of “Cyclic peptide tyrosine-tyrosine conjugate coupled to glucagon-like peptide 1 (GLP-1) fusion peptide and its use”. Technical Field

[0002] The present invention generally relates to novel glucagon-like peptide 1 (GLP-1) fusion peptide coupled cyclic peptide tyrosine-tyrosine (PYY) conjugates, which are modulators of neuropeptide Y2 receptor and GLP-1 receptor. The present invention also relates to pharmaceutical compositions and methods of use thereof. The novel GLP-1 fusion peptide coupled cyclic PYY conjugates can be used to prevent, treat or improve diseases and disorders such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia, etc. Background Art

[0003] The neuropeptide Y (NPY) receptor is activated by a group of closely related peptide agonists known as the "NPY family," which have different affinities for each receptor subtype. NPY, peptide tyrosine-tyrosine (PYY) and pancreatic polypeptide (PP), all 36 amino acids in length, are agonists of the NPY receptor family. NPY is a neurotransmitter that is synthesized, co-stored, and released with norepinephrine and epinephrine. NPY is one of the most abundant and widely distributed peptides in the central nervous system (CNS) of humans and rodents, and is expressed in areas of the brain associated with feeding and stress. In the peripheral nervous system, neurons containing NPY are primarily sympathetic. PYY is primarily synthesized and released by enteroendocrine cells. NPY is produced by cleavage of NPY and PYY by the endothelial serine protease, dipeptidyl peptidase IV (DPP-IV). 3-36 and PYY 3-36 , which is a selective ligand for the Y2 and Y5 subtypes of the NPY receptor family. PP is found primarily in pancreatic islet cells, which are distinct from those that store insulin, glucagon, or somatostatin.

[0004] Five different NPY receptors have been discovered to date, four of which are understood to be relevant to human physiology. Receptors Y1, Y2, and Y5 preferentially bind NPY and PYY, while the Y4 receptor preferentially binds PP. The Y2 and Y5 receptors are also efficiently activated by NPY3-36 and PYY3-36. In general, ligands of the NPY family have variable selectivity for each of the NPY receptor subtypes, with PYY3-36 previously reported to have moderate to robust selectivity for the Y2 subtype. Each of these receptors is coupled to pertussis toxin-sensitive Gαi to inhibit adenylate cyclase.

[0005] PYY is secreted from endocrine L-cells in response to food, and especially after ingestion of fat. 1-36 Dominant in the fasting state, with PYY 3-36 It is the major form present after a human meal, with plasma concentrations inversely correlated with the number of calories consumed. 3-36 Reduces food intake in humans, monkeys, rats, rabbits, and mice (Batterham et al., Nature 418(6898):650-4 (2002); Batterham et al., N Engl J Med 349(10):941-8 (2003); Challis et al., Biochem Biophys Res Commun 311(4):915-9 (2003)). PYY 3-36 The anorectic effects of PYY are thought to be Y2-mediated based on preferential binding at this receptor and loss of feeding function in Y2-deficient mice (Batterham et al., Nature 418(6898):650-4 (2002)). 3-36 Intra-arcuate injection of PYY reduces food intake in rats and mice (Batterham et al., Nature 418(6898):650-4 (2002)), suggesting that engagement of the hypothalamic Y2 receptor may mediate these effects. Acute effects on food intake have also been shown to translate into dose-dependent effects on body weight in ob / ob mice, DIO mice, and Zuckerfa / fa mice (Pittner et al., Int J Obes Relat Metab Disord 28(8):963-71 (2004)). In addition, PYY 3–36 It has also been shown to improve insulin-mediated glucose disposal and insulin sensitivity in DIO rodents (Vrang et al., Am J Physiol Regul Integr Comp Physiol 291(2):R367-75 (2006)). Bariatric surgery results in increased circulating PYY immunoreactivity (le Roux et al., Ann Surg 243(1):108-14 (2006)), which appears to play a role in postoperative weight loss.

[0006] Given its role in controlling appetite and food intake as well as its antisecretory and absorptive effects in the mammalian gastrointestinal tract, PYY 3-36 It is effective in treating obesity and related conditions as well as many gastrointestinal diseases. 3-36Its therapeutic utility as a treatment agent itself is limited by its rapid metabolism and resulting short circulation half-life (Torang et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 310: R866-R874 (2016)).

[0007] Activation of the incretin hormone GLP-1 and its receptor (GLP1R) has a range of beneficial effects on human glucose metabolism and energy balance, including stimulation of glucose-dependent insulin secretion (Kreymann et al., Lancet 2:1300-1304 (1987)), and inhibition of glucagon secretion (Gutniak et al., N Engl J Med 326:1316-1322 (1992)), gastric emptying (Wettergren et al., Digestive Diseases Sciences 38:665-673 (1993)), and food intake (Flint et al., JCI 101:515-520 (1998)). GLP1R agonists have also been shown to significantly reduce the risk of cardiovascular and microvascular outcomes (Marso et al., N Engl J Med 375(4):311-322 (2016)) and renal disease (Mann et al., N Engl J Med 377(9):839-848 (2017)) in patients with type 2 diabetes. GLP-1 is characterized by a short half-life, which makes the use of GLP-1 as a potential therapeutic agent impractical. The circulating half-life is limited by both proteolytic susceptibility from dipeptidyl peptidase IV (Zhu, L. et al., JBC 278:22418-22423 (2003)) and neutral endopeptidase (Hupe-Sodmann et al., Regul Pept 58(3):149-156 (1995)) and renal filtration (Ruiz-Grande et al., Can J Physiol Pharmacol 68(12):1568-1573 (1990)).

[0008] Therefore, it is expected that the 3-36 PYY analogs or derivatives thereof and / or GLP-1 analogs or derivatives thereof with improved metabolic stability and pharmacokinetic profiles and / or GLP-1. Such derivatives with a prolonged half-life in vivo will provide Y2 and / or GLP-1 receptor modulation with a greater duration of action, making them useful as therapeutic agents for subjects in need of such modulation.

[0009] The above discussion is presented merely to provide a better understanding of the nature of the problems faced in the art and should not be construed in any way as an admission of prior art, nor should citation of any reference herein be construed as an admission that such reference constitutes "prior art" to the present application. Summary of the invention

[0010] In one general aspect, the present invention relates to novel glucagon-like peptide 1 (GLP-1) fusion-coupled cyclic peptide tyrosine-tyrosine (PYY) conjugates that are modulators of the neuropeptide Y2 receptor and the GLP-1 receptor.

[0011] Provided herein are conjugates comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises a GLP-1 or GLP-1 variant peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, wherein optionally the first linker is absent.

[0012] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof:

[0013]

[0014] in

[0015] p is 0 or 1;

[0016] m is 0, 1, 2, 3, 4 or 5;

[0017] n is 1, 2, 3 or 4;

[0018] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0019] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0020] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0021] Z4 is K, A, E, S or R;

[0022] Z7 is A or K;

[0023] Z9 is G or K;

[0024] Z 11 is D or K;

[0025] Z 22 A or K;

[0026] Z23 is S or K;

[0027] Z 26 A or H;

[0028] Z 30 L, W or not present;

[0029] The premise is that only when q is 1, Z 30 does not exist;

[0030] Z 34 for

[0031] Z 35 for

[0032] Wherein the derivative is a compound of formula I modified by one or more processes selected from amidation, acylation and pegylation.

[0033] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof, wherein:

[0034] p is 0 or 1;

[0035] m is 0, 1, 2, 3, 4 or 5;

[0036] n is 1, 2, 3 or 4;

[0037] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0038] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-,

[0039] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0040] Z4 is K, A, E, S or R;

[0041] Z7 is A or K, wherein the amino side chain of K is optionally substituted by

[0042] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0043] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0044] Z9 is G or K, wherein the amino side chain of K is optionally substituted by

[0045] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0046] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0047] Z 11 is D or K, wherein the amino side chain of K is optionally substituted by

[0048] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0049] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0050] Z 22 is A or K, wherein the amino side chain of K is optionally substituted by

[0051] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0052] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0053] Z 23 is S or K, wherein the amino side chain of K is optionally substituted by

[0054] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0055] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0056] Z 26 A or H;

[0057] Z 30 is L;

[0058] Z 34 for and

[0059] Z 35 for

[0060] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof, wherein:

[0061] p is 0 or 1;

[0062] m is 0, 1, 2, 3 or 5;

[0063] n is 1, 2 or 4;

[0064] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q can be 1;

[0065] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0066] Z4 is K, A, E, S or R;

[0067] Z7 is A or K, wherein the amino side chain of K is substituted by

[0068] -C(O)CH2Br,

[0069] Z9 is G or K, wherein the amino side chain of K is substituted by

[0070] -C(O)CH2Br,

[0071] Z 11 is D or K, wherein the amino side chain of K is substituted by

[0072] -C(O)CH2Br,

[0073] Z 22 is A or K, wherein the amino side chain of K is substituted by

[0074] -C(O)CH2Br,

[0075] Z 23 is S or K, wherein the amino side chain of K is substituted by

[0076] -C(O)CH2Br,

[0077] Z 26 is A or H,

[0078] Z 30 For L,

[0079] Z 34 for

[0080] Z 35 for

[0081] In certain embodiments, the cyclic PYY peptide is selected from SEQ ID NO: 1-54, or a pharmaceutically acceptable salt thereof. In certain embodiments, the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34.

[0082] In certain embodiments, the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide. 11 , Z 22 and Z 23 Only one of the two is a lysine, and the lysine is covalently linked to a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide.

[0083] In certain embodiments, the GLP-1 peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from SEQ ID NOs: 56-59. In certain embodiments, the first linker peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from SEQ ID NOs: 60-83. In certain embodiments, the hinge-Fc region peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from SEQ ID NOs: 84-90. In certain embodiments, the second linker peptide of the GLP-1 fusion peptide comprises an amino acid sequence selected from SEQ ID NOs: 91-112. In certain embodiments, the second linker peptide comprises an amino acid sequence of SEQ ID NOs: 93, 94, 95, 106 or 111.

[0084] The present invention also provides a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and wherein the cyclic PYY peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33, or 34. In certain embodiments, the GLP-1 fusion peptide comprises an amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 136. In certain embodiments, a cysteine ​​residue between amino acid residues 287 to 289 of SEQ ID NO: 113 or SEQ ID NO: 136, preferably cysteine ​​residue 288 of SEQ ID NO: 113 or SEQ ID NO: 136, is covalently linked directly or via a chemical linker to a lysine residue at residue 7, 9, 11, 22, or 23 of the cyclic PYY peptide, preferably lysine residue 11 of the cyclic PYY peptide.

[0085] The present invention also provides a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the conjugate comprises a sequence selected from SEQ ID NOs: 225-262, or a pharmaceutically acceptable salt thereof.

[0086] The present invention also provides a method for preparing the conjugate of the present invention. The method comprises reacting an electrophile, preferably bromoacetamide or maleimide, introduced into the side chain of the cyclic PYY peptide, preferably the side chain of the lysine residue of the cyclic PYY peptide, with a sulfhydryl group of a cysteine ​​residue of the second linker peptide (e.g., a carboxyl terminal linker peptide) of the GLP-1 fusion peptide, thereby forming a covalent bond between the cyclic PYY peptide and the GLP-1 fusion peptide.

[0087] The present invention also provides a pharmaceutical composition comprising the conjugate of the present invention and a pharmaceutically acceptable carrier.

[0088] The present invention also provides a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and / or eczema. The method includes administering an effective amount of a pharmaceutical composition of the present invention to a subject in need thereof.

[0089] The present invention also provides a method of reducing at least one of food intake or body weight in a subject in need thereof. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0090] The present invention also provides a method for regulating Y2 receptor activity or GLP-1 receptor activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.

[0091] In certain embodiments, the pharmaceutical composition is administered via injection.

[0092] The present invention also provides a kit comprising a compound of the present invention, preferably also comprising liraglutide, and a device for injection.

[0093] The present invention also provides a method for preparing the pharmaceutical composition of the present invention, which comprises combining the conjugate with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0094] Other aspects, features and advantages of the present invention will be better understood from a reading of the following detailed description of the invention and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The foregoing summary and the following detailed description of the preferred embodiments of the present application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present application is not limited to the precise embodiments shown in the drawings.

[0096] Figure 1A to Figure 1B The in vitro human plasma stability of the GLP-1 fusion peptide portion is shown. Figure 1A The GLP-1 fusion peptides GF32 (SEQ ID NO 144) (▲), GF36 (148) (△), GF33 (145) (△) and GF36 (148) (△) were displayed in human plasma after incubation at 37°C for 7 days. GF39(151) Curve diagram of in vitro human plasma stability of GF34 (146) (⊙), GF35 (147) (●), GF37 (149) (○), GF40 (152) (◇) and dulaglutide control (◆). Figure 1B Shown are GF40 (SEQ ID NO: 152) (▲) and GF34 (SEQ ID NO: 146) (⊙) displayed in human plasma after incubation at 37°C for 7 days in vitro, and their corresponding GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates SEQ ID NO: 248 (△) and SEQ ID NO: 262 (○), and another GLP-1 fusion peptide GF41 (SEQ ID NO: 153) Plot of in vitro human plasma stability together with dulaglutide control (◆).

[0097] FIG. 2A to FIG. 2B Shown are food intake and body weight loss in DIO mice. Figure 2A and Figure 2B The food intake of DIO mice treated with dulaglutide, compound 1 (GF1 (SEQ ID NO: 113)) (0.3 nmol / kg) and compound 2 (SEQ ID NO: 225) (0.3 nmol / kg) is shown. Figure 2A ) and weight loss percentage ( Figure 2B ). The mice were administered vehicle (white circles), dulaglutide (white squares), compound 1 (black squares), or compound 2 (black circles) by subcutaneous injection, and body weight and food intake were recorded for 3 days.

[0098] FIG. 3A to FIG. 3B Shown are food intake and body weight loss in DIO mice. Figure 3A and Figure 3B The food intake of DIO mice treated with dulaglutide, compound 3 (GF24 (SEQ ID NO: 136)) (0.3 nmol / kg) and compound 4 (SEQ ID NO: 229) (0.3 nmol / kg) is shown. Figure 3A ) and weight loss percentage ( Figure 3B ). The mice were administered vehicle (white circles), dulaglutide (white squares), compound 3 (black squares), or compound 4 (black circles) by subcutaneous injection, and body weight and food intake were recorded for 3 days.

[0099] FIG. 4A to FIG. 4B Shown is the IPGTT of DIO mice. Figure 4A Shown is a graph of glucose levels over time in DIO mice treated with 0.3 nmol / kg and 1.0 nmol / kg of dulaglutide, Compound 4 (SEQ ID NO: 229), and Compound 2 (SEQ ID NO: 225). Figure 4B A graph of the net AUC relative to baseline for DIO mice treated with 0.3 nmol / kg and 1.0 nmol / kg of dulaglutide, compound 4 (SEQ ID NO: 229) and compound 2 (SEQ ID NO: 225) is shown. Mice were administered subcutaneously with vehicle (black circles), dulaglutide (white / black squares), compound 4 (white / black triangles) or compound 2 (white / black circles, dotted lines). All compounds were administered at 0.3 nmol / kg or 1.0 nmol / kg. After 18 hours of fasting, mice were administered 1 g / kg dextrose for intraperitoneal glucose tolerance test (IGPTT).

[0100] FIG. 5A to FIG. 5B Shown are food intake and body weight loss in DIO mice. Figure 5A and Figure 5B Shown is the food intake of DIO mice treated with dulaglutide, GF41 (SEQ ID NO: 153) (0.3 nmol / kg) and GF40 (SEQ ID NO: 152) (0.3 nmol / kg). Figure 5A ) and weight loss percentage ( Figure 5B ). Mice were dosed with vehicle (white circles), dulaglutide (white squares), SEQ ID NO: 153 (black squares), or SEQ ID NO: 152 (black circles) by subcutaneous injection, and body weight and food intake were recorded for 3 days.

[0101] FIG. 6A to FIG. 6B Shown are food intake and body weight loss in DIO mice. Fig. 6A and Figure 6B Figure 2 shows the food intake of DIO mice treated with dulaglutide and GF19 (SEQ ID NO: 131) (0.3 nmol / kg). Fig. 6A ) and weight loss percentage ( Figure 6B ). Mice were dosed with vehicle (white circles), dulaglutide (white squares), or SEQ ID NO: 131 (black squares) by subcutaneous injection, and body weight and food intake were recorded for 3 days.

[0102] FIG. 7A to FIG. 7B Shown is the IPGTT of DIO mice. Fig. 7A Shown is a graph of glucose levels over time in DIO mice treated with 0.3 nmol / kg of dulaglutide, Compound 4 (SEQ ID NO: 229), and Compound 3 (GF24 (SEQ ID NO: 136)). Figure 7B A graph of net AUC versus baseline for DIO mice treated with 0.3 nmol / kg of dulaglutide, compound 4 (SEQ ID NO: 229), and compound 3 (GF24 (SEQ ID NO: 136)) is shown. Mice were dosed subcutaneously with vehicle (black circles), dulaglutide (white squares), compound 4 (white circles), or compound 3 (black squares, dashed line). All compounds were dosed at 0.3 nmol / kg. After an 18-hour fast, mice were administered 1 g / kg dextrose for an intraperitoneal glucose tolerance test (IGPTT).

[0103] FIG. 8A to FIG. 8B Shown is an exposure response analysis of compound 4 (SEQ ID NO: 229) or dulaglutide on food intake and body weight changes after a single dose to DIO mice. Fig. 8A and Figure 8B The percentage change in food intake is shown ( Fig. 8A ) and percentage change in body weight ( Figure 8B ). The open squares represent the observed individual data for dulaglutide. The solid squares represent the observed individual data for compound 4. The curves represent the best fit curves based on nonlinear regression of exposure response for dulaglutide (grey curve) and compound 4 (black curve). The grey vertical line represents the mean (± standard deviation) day 3 HGE concentration of dulaglutide at 0.3 nmol / kg for all analyzed studies: ≈0.32 (±0.15) nM.

[0104] FIG. 9A to FIG. 9BExposure-response analysis of the effects of Compound 4 (black symbols) or dulaglutide (white squares) on caloric intake in overweight cynomolgus monkeys on days 9 (pre-dose) and 12 (pre-dose) after repeated (4, Q3D) subcutaneous (SC) dosing is shown. Fig.9A ) and LC-MS / MSHE determination ( Fig. 9B ) both measure active GLP-1 concentration. The curves represent the best fit curves based on nonlinear regression of exposure response of dulaglutide (grey curve) and compound 4 (black curve). Data from compound 4 dosed at 0.0074 mg / kg are not included in the exposure response analysis, but are only included in the curve graph. The daily caloric intake change% was calculated relative to the baseline, which was defined as the average from day -2 to day 0.

[0105] FIG. 10A to FIG. 10B Shown is the food intake (FI) (in grams) of DIO mice treated with vehicle, dulaglutide (0.3 nmol / kg), or SEQ ID NO: 140 (0.3 nmol / kg) ( Fig. 10A ) and body weight (BW) changes (% Fig. 10B ). One day before the start of the study, DIO mice were weighed and grouped by body weight. Mice were administered vehicle (white circles), dulaglutide (white squares), or SEQ ID NO 140 (black squares) by subcutaneous injection (2 mL / kg), and BW and FI were recorded for the next 3 days.

[0106] FIG. 11A to FIG. 11B Shown is the food intake (FI) (in grams) of DIO mice treated with vehicle, dulaglutide (0.3 nmol / kg), SEQ ID NO: 131 (0.3 nmol / kg), or SEQ ID NO: 251 (0.3 nmol / kg) ( Fig.11A ) and body weight (BW) changes (% Fig. 11B ). One day before the start of the study, DIO mice were weighed and grouped by body weight. Mice were administered subcutaneous injection (2 mL / kg) of vehicle (white circles), dulaglutide (white squares), SEQ ID NO 131 (black squares), or SEQ ID NO 251 (black circles), and BW and FI were recorded over the next 3 days.

[0107] Fig.12 A graph showing the results of a human GLP-1R primary screening assay comparing a GLP-1 peptide (SEQ ID NO: 58) with first linker peptides of varying lengths (N-terminal linker peptide) is shown. SEQ ID NO: 171 comprising the longest first linker peptide (SEQ ID NO: 67) is the most effective, followed by GLP-1 fusion peptides with shorter first linker peptides, i.e., SEQ ID NO 169 (▲) with first linker peptide SEQ ID NO 64 and SEQ ID NO 168 (■) with first linker peptide SEQ ID NO 74. SEQ ID NO 167 without an intervening first linker peptide between the GLP peptide and hinge-Fc peptide (●) is the least effective. GLP-1 peptide (◆) is included as an assay control. DETAILED DESCRIPTION

[0108] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0110] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0111] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include the value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0112] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0113] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0114] It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, the use of "about," "approximately," "substantially," and similar terms herein indicates that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without changing the least significant digits.

[0115] In two or more nucleic acid or polypeptide sequences (e.g., GLP-1 peptide, linker peptide, hinge-Fc region peptide, cyclic PYY 3-36 In the context of peptide sequences, the terms "same" or "percent identity" refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms in accordance with the present disclosure or by visual inspection using methods known in the art.

[0116] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the specified program parameters.

[0117] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by using the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by searching the similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988). 85:2444 (1988)), by algorithms (GAP, BESTFIT, FASTA and TFASTA, computerized implementations of the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Laboratory Protocols, a joint venture of Greene Publishing Associates and John Wiley & Sons, Inc. (1995 supplement) (Ausubel)).

[0118] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0119] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0120] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0121] In the context of the methods of the present invention, the term "administering" means a method of therapeutically or prophylactically preventing, treating or ameliorating the syndromes, disorders or diseases described herein by using a conjugate or compound of the present invention, or a form, composition or medicament thereof. Such methods include administering an effective amount of the conjugate, compound, form, composition or medicament thereof at different times during the course of treatment, or administering simultaneously in a combined form. The methods of the present invention should be understood to encompass all known therapeutic treatment regimens.

[0122] The term "effective amount" means the amount of an active conjugate, compound or agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, including the prevention, treatment or amelioration of the syndrome, disorder or disease being treated, or the prevention, treatment or amelioration of a symptom of the syndrome, disorder or disease being treated.

[0123] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0124] As used herein, the term "coupling" refers to the binding or connection of two or more objects together. When referring to chemical or biological compounds, coupling can refer to a covalent connection between two or more chemical or biological compounds. As a non-limiting example, the glucagon-like peptide 1 (GLP-1) fusion peptide of the present invention can be coupled to a cyclic PYY peptide of interest to form a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate. In certain embodiments, the GLP-1 fusion peptide of the present invention can be covalently coupled to the cyclic PYY peptide of the present invention through at least one linker. The GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate can be formed by a specific chemical reaction designed to conjugate the GLP-1 fusion peptide to the cyclic PYY peptide. For example, the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate can be formed by a conjugation reaction. The conjugation reaction can, for example, include reacting an electrophilic group (e.g., bromoacetamide or maleimide) with a sulfhydryl group of a cysteine ​​residue on a peptide of interest (e.g., a GLP-1 fusion peptide). The electrophilic group can be introduced, for example, on the side chain of an amino acid residue of the cyclic PYY peptide. The reaction of the electrophilic group with a sulfhydryl group results in the formation of a covalent thioether bond.

[0125] As used herein, the term "peptide linker" or "linker peptide" refers to a chemical module comprising one or more amino acids that connects the GLP-1 peptide to the hinge-Fc region peptide or connects the hinge-Fc region peptide to the cyclic PYY peptide to form a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate. The GLP-1 fusion peptide may, for example, comprise a first linker peptide and a second linker peptide.

[0126] As used herein, the term "chemical linker" refers to a chemical moiety that does not comprise any amino acids that connect the cyclic PYY peptide to the GLP-1 fusion peptide. In certain embodiments, the cyclic PYY peptide comprises a chemical linker. The chemical linker of the cyclic PYY peptide may, for example, include, but is not limited to, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a linker comprising an acyl group, a mixed linker consisting of PEG and an embedded heterocycle, and a hydrocarbon chain. The chemical linker may, for example, be first covalently linked to the cyclic PYY peptide and then covalently linked to the GLP-1 fusion peptide, preferably covalently linked to a second linker peptide of the GLP-1 fusion peptide.

[0127] As used herein, the term "conjugate" refers to a peptide (e.g., a GLP-1 fusion peptide) covalently coupled to another pharmaceutically active moiety (e.g., a cyclic PYY peptide). The term "conjugated to" means that the peptide of the present invention is covalently bonded or covalently linked to another pharmaceutically active moiety, preferably a therapeutic peptide, directly or indirectly via a linker. As a non-limiting example, the peptide may be a GLP-1 fusion peptide of the present invention, and the other pharmaceutically active moiety may be a therapeutic peptide, such as a cyclic PYY peptide of interest.

[0128] The peptide sequences described herein are written according to the usual convention, wherein the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, unless otherwise specifically indicated, it is the L form of the amino acid represented.

[0129] Glucagon-like peptide 1 (GLP-1) fusion peptide

[0130] In one overall aspect, the present invention relates to a glucagon-like peptide 1 (GLP-1) fusion peptide. The GLP-1 fusion peptide comprises a GLP-1 or GLP-1 variant peptide, a first linker peptide (e.g., an amino (N)-terminal linker), a hinge-Fc region peptide, and a second linker peptide (e.g., a carboxyl (C)-terminal linker).

[0131] Glucagon-like peptide 1 or GLP-1 variant peptide

[0132] Glucagon-like peptide 1 (GLP-1) is an insulin secretagogue synthesized in the intestine and released in response to food ingestion. It is secreted primarily in two forms: GLP-1-(7-37) and GLP-1-(7-36)NH2, both of which bind to specific GLP-1 receptors (GLP-1R) on pancreatic β cells and enhance glucose-stimulated insulin secretion.

[0133] Many GLP-1 analogs and derivatives are known and may be referred to herein as "GLP-1 variants". These GLP-1 variant peptides may include exendins, which are peptides found in the venom of the Gila monster. These exendins have sequence homology with native GLP-1 and can bind to the GLP-1 receptor and initiate the active signal transduction cascade associated with GLP-1 (7-37).

[0134] GLP-1 and GLP-1 variant peptides have been shown to act in a variety of ways, including but not limited to stimulation of insulin release, reduction of glucagon secretion, inhibition of gastric emptying, and enhancement of glucose utilization.

[0135] GLP-1R belongs to the class B family of 7-transmembrane heterotrimeric G protein-coupled receptors and is expressed in a wide range of tissues including, but not limited to, pancreatic islets, heart, kidney, stomach, intestine, nodose ganglion neurons of the vagus nerve, and α-cells, β-cells, and δ-cells in several regions of the central nervous system (CNS), including the hypothalamus and brainstem. s , Gα q , Gα i and Gα o (Montrose-Rafizadeh et al., Endocrinology 140:1132-40 (1999); Hallbrink et al., Biochim Biophys Acta 1546:79-86 (2001)), leading to an increase in intracellular calcium, adenylate cyclase, and phospholipase C, and activation of PKA, PKC, PI-3K, Epac2, and MAPK signaling pathways (Drucker et al., PNAS 84:3434-8 (1987); Wheeler et al., Endrocrinology 133:57-62 (1993); and Holz et al., JBC 270:17749-57 (1995)).

[0136] Provided herein are GLP-1 fusion peptides comprising a first component, wherein the first component is a GLP-1 or GLP-1 variant peptide. As used herein, the terms "GLP-1 peptide," "GLP-1 variant peptide," "GLP-1 peptide variant," and "GLP-1 or GLP-1 variant peptide" are used interchangeably. The GLP-1 or GLP-1 variant peptide may comprise one of the sequences provided in Table 1. The GLP-1 or GLP-1 variant peptide sequence can be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency after chemical conjugation to a cyclic PYY peptide, (v) lack of serine xylosylation or potential for serine xylosylation, and (vi) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate (e.g., in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide are capable of agonist activity at the GLP-1 and Y2 receptors, respectively)).

[0137] The GLP-1 or GLP-1 variant peptide constituting the first component of the GLP-1 fusion peptide is intended to encompass peptides that have sufficient homology and functionality with natural GLP-1. The GLP-1 or GLP-1 variant peptide is designed to be able to bind to the GLP-1 receptor on the β-cells in the pancreas, thereby generating the same signal transduction pathway as when natural GLP-1 binds to the GLP-1 receptor on the β-cells in the pancreas and exhibiting the same or similar insulinotropic activity.

[0138] Table 1: Glucagon-like peptide 1 and its variants

[0139]

[0140] First linker peptide: amino terminal linker (N-terminal linker)

[0141] Provided herein are GLP-1 fusion peptides comprising a second component, wherein the second component is a first linker peptide (i.e., an amino terminal linker peptide). The first linker peptide may comprise one of the sequences provided in Table 2. The first linker peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, and (v) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate (e.g., in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide are capable of having agonist activity on the GLP-1 and Y2 receptors, respectively)).

[0142] In certain embodiments, the GLP-1 fusion peptide does not comprise a second component, wherein the second component is a first linker peptide.

[0143] Table 2: First linker peptide (N-terminal linker peptide)

[0144]

[0145] Hinge-Fc region peptide

[0146] In certain embodiments, the GLP-1 fusion peptide comprises a third component, wherein the third component is a hinge-Fc region peptide. In addition to FcRn receptor recycling, the hinge-Fc region peptide can also confer a longer circulation half-life to the therapeutic peptide by increasing the molecular weight and reducing glomerular filtration. In certain embodiments, the hinge-Fc region peptide can be derived from a human IgG4 Fc region. Compared with other IgG subtypes, the human IgG4 Fc region has a reduced ability to bind FcγR and complement factors. Preferably, the Fc region comprises a substituted human IgG4 Fc region with eliminated effector functions. Thus, the GLP-1 fusion peptide also comprises an Fc region having a modified human IgG4 Fc region containing one or more of the following substitutions: substitution of proline for glutamic acid at residue 233, substitution of alanine or valine for phenylalanine at residue 234, and substitution of alanine or glutamic acid for leucine at residue 235 (EU numbering, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U. S. Pat. of Health and Human Services, Bethesda, Md., NIH Publication No. 91-3242 (1991). Removal of the N-linked glycosylation site in the IgG4 Fc region by substitution of Ala for Asn at residue 297 (EU numbering) is another way to ensure elimination of residual effector activity.

[0147] In certain embodiments, the GLP-1 fusion peptide of the present invention exists as a monomer or dimer. In a preferred embodiment, the GLP-1 fusion peptide exists as a dimer. In certain embodiments, where the GLP-1 fusion peptide exists as a dimer, the dimer is a homodimer, i.e., the dimer contains two GLP-1 fusion peptides with the same sequence. In certain embodiments, where the GLP-1 fusion peptide exists as a dimer, the dimer is a heterodimer, i.e., the dimer contains two GLP-1 fusion peptides with different sequences.

[0148] Preferably, the GLP-1 fusion peptide of the present invention exists as a dimer linked together by disulfide bonds and various non-covalent interactions. Therefore, the Fc portion that can be used for the antibody of the present invention can be a human IgG4 Fc region containing substitutions, such as a substitution of serine to proline at position 228 (EU numbering), which stabilizes the formation of heavy chain dimers and prevents the formation of half IgG4 Fc chains. In certain embodiments, the natural amino acid sequence at the N-terminus of the hinge disulfide bond may be included. The x-ray crystal structure of human IgG4 shows that these amino acids can form a structure that tends to set the upstream structure in the opposite orientation in space. This tendency is conducive to maintaining the spatial separation of upstream elements such as GLP-1 fusion peptides. In other embodiments, the natural amino acids at the N-terminus of the hinge disulfide bond can be omitted.

[0149] In certain embodiments, a hybrid human IgG2a hinge fused to human PAAFc was used to explore the effects on the potency and stability of the GLP-1 fusion peptide.

[0150] In another embodiment, the C-terminal Lys residue in the heavy chain is removed, as is common in recombinantly produced monoclonal antibodies.

[0151] The hinge-Fc region peptide may comprise one of the sequences provided in Table 3. The hinge-Fc region peptide sequence may be selected based on at least one of the following criteria: (i) in vitro stability, (ii) in vitro potency, and (iii) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate (e.g., in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide are capable of agonist activity at the GLP-1 and Y2 receptors, respectively)).

[0152] Table 3: Hinge-Fc region peptides

[0153]

[0154]

[0155] Also provided herein is a hinge-Fc region platform peptide, wherein the hinge-Fc region platform peptide comprises a hinge-Fc region peptide, and at least one of a first linker peptide connected to the amino terminus of the hinge-Fc region peptide and a second linker peptide connected to the carboxyl terminus of the hinge-Fc region peptide. The hinge-Fc region peptide can, for example, be an amino acid sequence selected from SEQ ID NO: 84-90; the first linker peptide can, for example, be an amino acid sequence selected from SEQ ID NO: 60-83; and the second linker peptide can, for example, be an amino acid sequence selected from SEQ ID NO: 91-112. In certain embodiments, the hinge-Fc region peptide comprises SEQ ID NO: 84, and the first linker peptide comprises SEQ ID NO: 60. In certain embodiments, the hinge-Fc region peptide comprises SEQ ID NO: 84, and the second linker peptide comprises SEQ ID NO: 93, 94, 95, 106 or 111. In certain embodiments, the hinge-Fc region peptide comprises SEQ ID NO:84; the first linker peptide comprises SEQ ID NO:60; and the second linker peptide comprises SEQ ID NO:93, 94, 95, 106, or 111.

[0156] Second linker peptide: Carboxyl terminal linker (C-terminal linker)

[0157] Provided herein are GLP-1 fusion peptides comprising a fourth component, wherein the fourth component is a second linker peptide (i.e., a carboxyl terminal linker peptide). The second linker peptide may comprise one of the sequences provided in Table 4. The second linker peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, (v) conjugation yield, and (vi) properties of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate (e.g., in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide are capable of having agonist activity on the GLP-1 and Y2 receptors, respectively)).

[0158] In addition, the second linker peptide sequence can be selected based on its ability to undergo specific and efficient conjugation with the cyclic PYY peptide. In this context, specificity refers to the preference of conjugation at the engineered cysteine ​​residue in the C-terminal region of the second linker over conjugation at any other part of the fusion protein. Examples of strategies to maximize specificity include (i) introducing amino acids adjacent to the engineered cysteine ​​residues to increase the nucleophilicity of the sulfhydryl side chains and enhance their reactivity, (ii) including engineered cysteine ​​residues in amino acid sequences that have previously shown enhanced selectivity in thiol-electrophile conjugation, and (iii) the binding of amino acids to anionic or cationic side chains that electrostatically attract cationic or anionic disulfide bond reducing agents, respectively, to specifically release reactive thiols for conjugation. Examples of strategies to maximize conjugation efficiency include (i) introduction of anionic or cationic like charges at any part of the second linker peptide in order to induce mutual electrostatic repulsion of the second linker peptide and thus reduce disulfide bond formation between the engineered cysteine ​​residues, (ii) incorporation of amino acids that enhance linker rigidity, (iii) incorporation of amino acids that increase linker flexibility, and (iv) incorporation of amino acids with anionic or cationic side chains that tend to electrostatically facilitate efficient conjugation of the cyclic PYY peptide.

[0159] Table 4: Second linker peptide (C-terminal linker peptide)

[0160]

[0161] GLP-1 fusion peptide

[0162] Provided herein is a GLP-1 fusion peptide comprising a first component, a second component, a third component, and a fourth component as previously described, wherein the second component may optionally be absent. The first component is a GLP-1 or GLP-1 variant peptide, the second component is a first linker peptide, the third component is a hinge-Fc region peptide, and the fourth component is a second linker peptide. The GLP-1 fusion peptide may comprise one of the sequences provided in Table 5. The GLP-1 fusion peptide sequence may be selected based on at least one of the following criteria: (i) expression yield, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation, (v) conjugation yield, and (vi) properties of the cyclic PYY peptide conjugate coupled to the GLP-1 fusion peptide (e.g., in vivo stability and in vivo potency (i.e., whether the GLP-1 or GLP-1 variant peptide and the cyclic PYY peptide are able to have agonist activity on the GLP-1 receptor and the Y2 receptor, respectively)).

[0163] Table 5: GLP-1 fusion peptides

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Cyclic PYY peptide

[0183] PYY 3-36 PYY is an endogenous hormone secreted by L cells in the distal intestine that acts as an agonist of the Y2 receptor to inhibit food intake. Given its role in controlling appetite and food intake in the mammalian gastrointestinal tract and its antisecretory and pro-absorptive effects, PYY is 3-36 It is effective in treating obesity and related conditions as well as many gastrointestinal diseases. 3–36 Its therapeutic utility as a treatment agent itself is limited by its rapid metabolism and short circulating half-life. 3-36 conjugates that prolong PYY 3-36 The half-life of the peptide and reduced the metabolism of the peptide in the body.

[0184] In certain embodiments of the invention, the modified PYY 3-36 The peptide is a cyclic PYY peptide. The terms "cyclic PYY peptide", "cyclic PYY 3-36 Analogs" and "cyclic PYY 3-36 "Peptide analogs" are used interchangeably. Examples of cyclic PYY peptides that can be used in the conjugates of the present invention are described in U.S. patent application Ser. No. 15 / 794,231, filed on Oct. 26, 2017, and U.S. patent application Ser. No. 15 / 794,171, filed on Oct. 26, 2017, the contents of both applications are hereby incorporated by reference in their entirety.

[0185] As used herein, the term "NTSC-PYY" is intended to describe the N-terminal to side chain cyclic analog of PYY.

[0186] The peptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, unless otherwise specifically indicated, it is the L form of the amino acid represented. For ease of describing the molecules of the invention, conventional and unconventional abbreviations for the various amino acids (both single and three letter codes) and functional moieties are used. These abbreviations will be familiar to those skilled in the art, but for the sake of clarity are listed below: A = Ala = alanine; R = Arg = arginine; N = Asn = asparagine; D = Asp = aspartic acid; βA = βAla = β-alanine; C = Cys = cysteine; hC = hCys = homocysteine; E = Glu = glutamate; Q = Gln = glutamate; G = Gly = glycine; H = His = histidine; I = Ile = isoleucine; L = Leu = leucine; K = Lys = lysine; Nle = norleucine; F = Phe = phenylalanine; P = Pro = proline; S = Ser = serine; T = Thr = threonine; W = Trp = tryptophan; Y = Tyr = tyrosine and V = Val = valine.

[0187] For convenience, the amino acid residue numbering convention used to name the NTSC-PYY peptides of the present invention follows hPYY 3-36 The specific amino acid substitutions that have been introduced into the NTSC-PYY peptide relative to hPYY 3-36 The native residue at the corresponding position in hPYY is indicated by the appropriate amino acid code, followed by the position of the substitution. Thus, "S4" in the NTSC-PYY peptide refers to a peptide in which serine has been substituted for hPYY. 3-36 Similarly, "hC31" in the NTSC-PYY peptide refers to a peptide in which homocysteine ​​has been substituted for hPYY 3-36Following this convention, additional amino acid substitutions occurring within the NTSC-PYY peptide are described and will likewise be recognized by those skilled in the art.

[0188] For convenience, the naming convention for the NTSC-PYY peptides of the present invention combines the amino acid residues involved in the cycle together with the linking group between them, starting from the N-terminal residue involved in the cycle, in a left-to-right direction. In all cases, the N-terminal amino acid residue of the cycle is linked to the linking group with its α-amino functional group, which is in turn linked to the amino acid side chain residue at position 31 of the NTSC-PYY peptide. Therefore, "cyclo-(I3-m-COPhCH2-hC31)" is used to describe the cycle of the NTSC-PYY peptide, in which the α-amino functional group of Ile3 is acylated with an m-methylbenzoic acid residue, the methyl group of which is further linked to the side chain of the hCys31 residue through a thioether bond. Similarly, “cyclo-(K4-CO(CH2)2NHCOCH2-hC31)” is used to describe a cycle of the NTSC-PYY peptide in which the native Ile3 residue has been deleted and the α-amino function of its (now N-terminal) lys4 is acylated by a 3-acetamidopropionyl group, the acetamido methylene carbon of which is linked to the side chain of the hCys31 residue via a thioether bond.

[0189] Lysine residues can bind to hPYY 3-36 Various positions of the sequence can be modified to provide convenient functional handles for further derivatization. Lysine residues can be modified to be indirectly coupled to the GLP-1 fusion peptide. In the indirect coupling with the GLP-1 fusion peptide, the lysine residue can be modified to contain a chemical linker that will allow the cyclic PYY peptide to be coupled to the GLP-1 fusion peptide. Those skilled in the art will recognize that related orthologs can also be used effectively in this way and are contemplated herein.

[0190] The term "K(PEG24-AcBr)" refers to a lysinyl residue whose side chain ε-amino group has been acylated via its 1-carboxylic acid function with N-bromoacetyl-75-amino-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetraoxapentadecanoic acid.

[0191] The term "K(PEG12-AcBr)" refers to a lysinyl residue whose side chain ε-amino group has been acylated via its 1-carboxylic acid function with N-bromoacetyl-39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodeca-triacontadecanoic acid.

[0192] The term "K(PEG6-AcBr)" refers to a lysinyl residue whose side chain ε-amino group has been acylated via its 1-carboxylic acid function with N-bromoacetyl-3-[(17-amino-3,6,9,12,15-pentaoxaheptadecan-1-yl)oxy]propionic acid.

[0193] The term "K(PEG8-triazolyl-CH2CH2CO-PEG4-AcBr)" refers to a lysinyl residue whose side chain ε-amino group has been acylated via its 1-carboxylic acid function with 27-[4-[2-[3-[2-[2-[3-(N-bromoacetylamino)propoxy]ethoxy]ethoxy]propylaminocarbonyl]ethyl]tetrazol-1-yl]-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid.

[0194] Many of the compounds / conjugates of the invention incorporate a reduced amide bond between the C-terminal residue Y36 of the sequence and its adjacent residue R35. This reduced amide bond is represented by the term "psi-(R35, Y36)".

[0195] Various amino acid residues comprising certain sequences of the invention contain methylated α-amino groups. Thus, the terms "N-Me-Q34" or "N-Me-R35" refer to the α-N-methylated glutamine at position 34 of the sequence and the α-N-methylated arginine at position 35 of the sequence, respectively.

[0196] The term "N-Me-Q34,psi-(R35,Y36)" in the sequence description refers to a sequence comprising both an alpha-methylglutamine residue at position 34 and a reduced amide bond between residues R35 and Y36.

[0197] Similarly, the term "N-Me-R35,psi-(R35,Y36)" in the sequence description refers to a sequence comprising both an α-methylarginine residue at position 35 and a reduced amide bond between this residue and Y36.

[0198] Examples of cyclic PYY peptides are provided in Table 6.

[0199] Table 6: Cyclic PYY peptides

[0200]

[0201]

[0202]

[0203] Also provided herein are N-terminal to side chain cyclic analogs of PYY that exhibit similarity to human PYY. 3-36 (hPYY 3-36). As an example of a method for determining sequence identity between two analogs, two peptides are

[0204]

[0205] (SEQ ID NO: 1) and hPYY 3-36

[0206] (SEQ ID NO: 55) was compared. 3-36 The sequence identity of hPYY is calculated by subtracting the number of different residues (i.e., the number of identical residues aligned) from the total number of aligned residues and dividing by 3-36 The total number of residues in is given. In this example, the different residues are D11, which has been exchanged for substituted K11, then V31, which has been exchanged for hC31, and finally R35 has been decarbonylated. Therefore, in the example, the sequence identity is (34-3) / 34X100.

[0207] Conjugate

[0208] In another overall aspect, the present invention relates to a conjugate comprising a GLP-1 fusion peptide of the present invention covalently conjugated to a cyclic PYY peptide in a site-specific manner, such that the GLP-1 fusion peptide-coupled cyclic PYY peptide has an extended / increased half-life compared to a single GLP-1 or GLP-1 variant peptide or cyclic PYY peptide. The present invention also relates to pharmaceutical compositions and methods of use thereof. The conjugates can be used to prevent, treat or ameliorate diseases or disorders such as obesity, type 2 diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and eczema, among others.

[0209] In certain embodiments, the GLP-1 fusion peptide of the present invention comprises at least one cysteine ​​residue capable of being conjugated to a cyclic PYY peptide. In certain embodiments, the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274. In certain embodiments, the GLP-1 fusion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113 or 136. In certain embodiments, the at least one cysteine ​​residue is contained in the second linker peptide of the GLP-1 fusion peptide. In certain embodiments, the cysteine ​​residue is located between residues 287 and 289 of the GLP-1 fusion peptide of SEQ ID NO: 113 or SEQ ID NO: 136, preferably the cysteine ​​residue is located at residue 288 of the GLP-1 fusion. In certain embodiments, the GLP-1 fusion peptide is covalently linked to a lysine residue at residue 7, 9, 11, 22 or 23 of the cyclic PYY peptide, preferably at lysine residue 11. In certain embodiments, the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide directly, or indirectly via a chemical linker on the cyclic PYY peptide.

[0210] In certain embodiments, the cyclic PYY peptide may comprise a chemical linker. The chemical linker may be chemically modified (e.g., an electrophilic group may be added to the linker) to allow the GLP-1 fusion peptide to be conjugated to the cyclic PYY chemical linker. The chemical linker may, for example, include, but is not limited to, a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a linker comprising an acyl group, a mixed linker consisting of PEG and an embedded heterocycle, or a hydrocarbon chain. The PEG linker may, for example, comprise 2-24 PEG units.

[0211] Methods for conjugating the GLP-1 fusion peptide of the present invention to the cyclic PYY peptide of the present invention are known in the art. In short, the GLP-1 fusion peptide of the present invention can be reduced with a reducing agent (e.g., 1,3,5-triaza-7-phosphaadamantane (PTA)), purified (e.g., by desalting chromatography) and conjugated with a cyclic PYY peptide (e.g., by providing a reduced GLP-1 fusion peptide under conditions that allow conjugation). During the conjugation reaction, the electrophilic leaving group of the cyclic PYY peptide is displaced, and a covalent bond is formed between the GLP-1 fusion peptide and the cyclic PYY peptide to form a cyclic PYY peptide conjugate coupled to the GLP-1 fusion peptide. After the conjugation reaction, the conjugate can be purified by ion exchange chromatography or hydrophobic interaction chromatography (HIC), and the final purification step is protein A adsorption. In certain embodiments, the GLP-1 fusion peptide of the present invention can be purified before reduction using the HIC method. For a more detailed description of the conjugation method, see, for example, Example 2.

[0212] Provided herein are conjugates comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, wherein optionally the first linker peptide is absent.

[0213] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof:

[0214]

[0215] in

[0216] p is 0 or 1;

[0217] m is 0, 1, 2, 3, 4 or 5;

[0218] n is 1, 2, 3 or 4;

[0219] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0220] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0221] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0222] Z4 is K, A, E, S or R;

[0223] Z7 is A or K;

[0224] Z9 is G or K;

[0225] Z 11 is D or K;

[0226] Z 22 A or K;

[0227] Z 23 is S or K;

[0228] Z 26 A or H;

[0229] Z 30 L, W, does not exist,

[0230] The premise is that only when q is 1, Z 30 does not exist;

[0231] Z 34 for

[0232] Z 35 for

[0233] Wherein the derivative is a compound of formula I modified by one or more processes selected from amidation, acylation and pegylation.

[0234] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof, wherein:

[0235] p is 0 or 1;

[0236] m is 0, 1, 2, 3, 4 or 5;

[0237] n is 1, 2, 3 or 4;

[0238] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0239] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0240] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0241] Z4 is K, A, E, S or R;

[0242] Z7 is A or K, wherein the amino side chain of K is optionally substituted by

[0243] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0244] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0245] Z9 is G or K, wherein the amino side chain of K is optionally substituted by

[0246] wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0247] Z 11 is D or K, wherein the amino side chain of K is optionally substituted by

[0248] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0249] -C(O)CH2I, -C(O)CH2Cl or -C(O)CH2Br;

[0250] Z 22 is A or K, wherein the amino side chain of K is optionally substituted by

[0251] wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0252] Z 23 is S or K; wherein the amino side chain of said K is optionally substituted by

[0253] wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0254] Z 26 A or H;

[0255] Z 30 L, W, does not exist, the premise is that only when q is 1, Z 30 does not exist;

[0256] Z 34 for

[0257] Z 35 for

[0258] wherein X is an electrophilic group, and Br, Cl or I of the electrophilic group of X is replaced in the conjugation reaction to form a cyclic PYY peptide conjugate coupled to a GLP-1 fusion peptide.

[0259] In certain embodiments, the cyclic PYY peptide is represented by Formula I or a derivative or pharmaceutically acceptable salt thereof, wherein:

[0260] p is 0 or 1;

[0261] m is 0, 1, 2, 3 or 5;

[0262] n is 1, 2 or 4;

[0263] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0264] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0265] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0266] Z4 is K, A, E, S or R;

[0267] Z7 is A or K, wherein the amino side chain of K is substituted by

[0268]

[0269] Z9 is G or K;

[0270] Z 11 is D or K, wherein the amino side chain of K is optionally substituted by

[0271]

[0272] -C(O)CH2Br, or

[0273] Z 22 is A or K, wherein the amino side chain of K is substituted by

[0274]

[0275] Z 23 is S or K, wherein the amino side chain of K is substituted by

[0276]

[0277] Z 26 A or H;

[0278] Z 30 is L;

[0279] Z 34 for

[0280] Z 35 for In which Br is replaced in the conjugation reaction to form a cyclic PYY peptide conjugate coupled to the GLP-1 fusion peptide.

[0281] In certain embodiments, the conjugate comprises a GLP-1 fusion peptide conjugated to a cyclic PYY peptide, wherein the cyclic PYY peptide is selected from SEQ ID NO: 1-54. In a preferred embodiment, the conjugate comprises a GLP-1 fusion peptide conjugated to a cyclic PYY peptide, wherein the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34.

[0282] In certain embodiments, the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide via a chemical linker on the lysine residue. The chemical linker may, for example, include a linker selected from C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain having 2 to 24 PEG units, a linker comprising an acyl group, or an alkyl chain comprising 2 to 10 carbon atoms.

[0283] The GLP-1 fusion peptide according to embodiments of the present invention may be conjugated to the cyclic PYY peptide at one or more amino acid positions of the cyclic PYY peptide, such as at amino acid residue 4, 7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30 or 31 of the cyclic PYY peptide using methods known in the art. The amino acid residue numbering follows hPYY 3-36 In certain embodiments, Z7, Z9, Z 11 , Z 22 and Z 23 In one preferred embodiment, only one of the two is lysine, and the lysine is covalently linked to a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide. In a preferred embodiment, the GLP-1 fusion peptide according to an embodiment of the present invention is conjugated to the cyclic PYY peptide at residue 11 of the cyclic PYY peptide, wherein residue 11 is lysine. In another preferred embodiment, an electrophile such as bromoacetamide or maleimide is introduced to the side chain of the cyclic PYY peptide such as the amino side chain of lysine at residue 11 of the cyclic PYY peptide, and the electrophile reacts site-specifically with the sulfhydryl group of the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, preferably the second linker peptide of the GLP-1 fusion peptide is SEQ ID NO: 93, 94, 95, 106 or 111, thereby forming a covalent bond between the cyclic PYY peptide and the GLP-1 fusion peptide. More preferably, the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34. In one embodiment, the electrophile is introduced directly onto the side chain of cyclic PYY. In another embodiment, the electrophile is introduced indirectly onto the side chain of cyclic PYY via a chemical linker.

[0284] In certain embodiments, the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, thereby reacting the reduced cysteine ​​residue with an electrophile. The azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

[0285] The present invention also provides a pharmaceutical composition comprising the conjugate of the present invention and further comprising a pharmaceutically acceptable carrier.

[0286] Non-limiting examples of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates are provided in Table 7.

[0287] Table 7: GLP-1 fusion peptide coupled cyclic PYY peptide conjugates

[0288]

[0289]

[0290]

[0291] Half-life extension

[0292] In addition to the GLP-1 fusion peptide, the conjugates of the present invention may be combined with one or more other moieties for extending the half-life of the pharmaceutically active moiety (e.g., cyclic PYY peptide), for example, via covalent interactions. Exemplary other half-life extension moieties include, but are not limited to, albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and fragments thereof, and the like. Additional half-life extension moieties that may be incorporated into the conjugates of the present invention include, for example, polyethylene glycol (PEG) molecules such as PEG5000 or PEG20,000, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) for the desired properties. These moieties may be directly fused to the protein scaffold coding sequence and may be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to connect these moieties to the recombinant and chemically prepared conjugates of the present invention.

[0293] A pegyl moiety, for example, can be added to a peptide molecule of the invention by attaching a cysteine ​​residue to the C-terminus of the molecule and linking the pegyl group to the cysteine ​​using well known methods.

[0294] The functionality of peptide molecules of the invention incorporating additional moieties can be compared by a variety of well-known assays. For example, the biological or pharmacokinetic activity of the therapeutic peptide of interest, alone or in the form of a conjugate according to the invention, can be determined and compared using known in vitro or in vivo assays.

[0295] Pharmaceutical composition

[0296] In another general aspect, the present invention relates to a pharmaceutical composition comprising the conjugates and compounds of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising the conjugates of the present invention and a pharmaceutically acceptable carrier. The conjugates and compounds of the present invention and compositions comprising them can also be used to manufacture medicaments for the therapeutic applications described herein.

[0297] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome capsule, or other material known in the art for use in pharmaceutical preparations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for the specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of the composition according to the present invention or the biological activity of the composition according to the present invention. According to the present disclosure, according to specific embodiments, any pharmaceutically acceptable carrier suitable for conjugated peptide pharmaceutical compositions can be used in the present invention.

[0298] Pharmaceutically acceptable acid salts / anionic salts used in the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etopate, esylate, fumarate, glucoheptonate, gluconate, glutamate, p-hydroxyacetamidophenylarsonic acid salt, hexylresorcinol salt, hydrazine, hydrobromide, Hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthenate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theochlorate, toluenesulfonate and triethiodide. Organic or inorganic acids also include, but are not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, isethionic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharinic acid or trifluoroacetic acid.

[0299] Pharmaceutically acceptable base / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine, or "TRIS"), ammonia, benzathine, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0300] In some embodiments of the present invention, a pharmaceutical formulation is provided, comprising a conjugate of the present invention in an amount of about 0.001 mg / ml to about 100 mg / ml, about 0.01 mg / ml to about 50 mg / ml, or about 0.1 mg / ml to about 25 mg / ml. The pharmaceutical formulation may have a pH of about 3.0 to about 10, such as about 3 to about 7, or about 5 to about 9. The formulation may also comprise at least one component selected from the group consisting of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and a surfactant.

[0301] The preparation of pharmaceutically active ingredients and pharmaceutically acceptable carriers is known in the art, such as Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulators, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present invention.

[0302] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation comprising water. The liquid formulation may comprise a solution, a suspension, an emulsion, a microemulsion, a gel, etc. The aqueous formulation typically comprises at least 50% w / w of water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w of water.

[0303] In one embodiment, the pharmaceutical composition can be formulated as an injection that can be injected, for example, via an injection device (eg, a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0304] In another embodiment, the pharmaceutical composition is a solid preparation, such as a freeze-dried or spray-dried composition, which can be used as is, or a solvent and / or diluent can be added by a physician or patient before use. Solid dosage forms can include tablets, such as compressed and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition can also be in the form of a sachet, dragee, powder, granule, lozenge, or powder, for example, for reconstitution.

[0305] Dosage forms may be immediate release, in which case they may contain a water soluble or dispersible carrier, or they may be delayed, sustained or modified release, in which case they may contain a water insoluble polymer that modulates the rate of dissolution of the dosage form in the gastrointestinal tract.

[0306] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0307] The pH in the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0308] In one embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, sodium dihydrogen phosphate, fumaric acid, glycine, glycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine and tris (hydroxymethyl) aminomethane and mixtures thereof. Buffers may exist alone or in aggregates with a concentration of about 0.01 mg / ml to about 50 mg / ml, such as about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the present invention.

[0309] In one embodiment of the invention, pharmaceutical composition comprises preservative.The non-limiting example of preservative comprises: benzethonium chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, 4-hydroxy butyl benzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin ether, o-cresol, meta-cresol, p-cresol, 4-hydroxy ethyl benzoate, imidourea, 4-hydroxy methyl benzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, 4-hydroxy propyl benzoate, sodium dehydroacetate, thimerosal and their mixture.Preservative can exist alone or in aggregate with about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml concentration exists.Comprising each pharmaceutical composition in these specific preservatives constitutes alternative embodiment of the present invention.

[0310] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of this embodiment include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), sugar alcohols (such as glycerol, 1,2-propylene glycol, propylene glycol), 1,3-propylene glycol and 1,3-butylene glycol), polyethylene glycol (e.g., PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars can be monosaccharides, disaccharides or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, amylopectin, dextrin, cyclodextrin, α and β-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is sugar alcohol, wherein the term "sugar alcohol" is defined as a C (4-8) hydrocarbon with at least one -OH group. The non-limiting example of sugar alcohol includes mannitol, sorbitol, inositol, galactitol, hexadecene, xylitol and arabitol. Isotonic agents can exist alone or in aggregates with a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific isotonic agents constitute alternative embodiments of the present invention.

[0311] In one embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. Chelating agents may exist alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the present invention.

[0312] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants and / or one or more protease inhibitors.

[0313] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or thioglycolic acid. The stabilizer may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the present invention.

[0314] In another embodiment of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble portion (hydrophilicity) and a fat-soluble and part (lipophilicity). Surfactant can be, for example, selected from anionic surfactants, cationic surfactants, nonionic surfactants and / or zwitterionic surfactants. Surfactant can exist alone or in aggregates with a concentration of about 0.1mg / ml to about 20mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the present invention.

[0315] In another embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present alone or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0316] Pharmaceutical composition of the present invention may include a certain amount of amino acid bases that are enough to reduce the aggregation formation of polypeptide during storage of the composition.Term " amino acid base " refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine) or their analogs.Any amino acid can exist in its free alkali form or its salt form.Any stereoisomer (i.e. L, D or their mixture) of amino acid base may be present.Amino acid base may exist alone or in combination with other amino acid bases with a concentration of about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml.The pharmaceutical composition comprising each of these specific amino acid bases constitutes an alternative embodiment of the present invention.

[0317] Pharmaceutically acceptable salts of the conjugates of the present invention include conventional non-toxic salts or quaternary ammonium salts formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetates, adipates, benzoates, benzenesulfonates, citrates, camphorates, dodecyl sulfates, hydrochlorides, hydrobromides, lactates, maleates, methanesulfonates, nitrates, oxalates, pivalates, propionates, succinates, sulfates and tartrates. Alkali salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine salts, and amino acids such as arginine. In addition, basic nitrogen-containing groups can be quaternized with, for example, alkyl halides.

[0318] The pharmaceutical compositions of the present invention can be administered in any manner that achieves its intended purpose. Examples include administration by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or ocular routes. Administration can be by oral route. Formulations suitable for parenteral administration include aqueous solutions of active conjugates (e.g., water-soluble salts), acidic solutions, alkaline solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes in water-soluble forms. In certain embodiments, the conjugates of the present invention are administered peripherally.

[0319] The present invention also encompasses a method for preparing a pharmaceutical composition, the method comprising mixing a pharmaceutically acceptable carrier with any of the conjugates of the present invention. In addition, the present invention includes a pharmaceutical composition prepared by mixing one or more pharmaceutically acceptable carriers with any of the conjugates of the present invention.

[0320] In addition, the conjugates of the present invention may have one or more polymorphs or amorphous crystalline forms, and are therefore intended to be included within the scope of the present invention. In addition, these conjugates can form solvates (i.e., hydrates) with, for example, water or with commonly used organic solvents. The term "solvate" as used herein means the physical association of the conjugate of the present invention with one or more solvent molecules. The physical association involves varying degrees of ionic bonding and covalent bonding, including hydrogen bonding. In some cases, the solvate will be able to separate when, for example, one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. The term "solvate" is intended to cover both solution phase solvates and separable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, etc.

[0321] The present invention is intended to include polymorphs and solvates of the conjugates of the present invention within its scope. Therefore, in the therapeutic methods of the present invention, the term "administering" should cover methods for treating, improving or preventing the syndromes, disorders or diseases described herein using the conjugates of the present invention or their polymorphs or solvates, which, although not specifically disclosed, will be clearly included within the scope of the present invention.

[0322] In another embodiment, the invention relates to a conjugate of the invention for use as a medicament.

[0323] The present invention includes within its scope prodrugs of the conjugates of the present invention. Generally speaking, such prodrugs will be functional derivatives of the conjugates, which can be easily converted into the desired conjugate in vivo. Therefore, in the methods of treatment of the present invention, the term "administering" should cover the use of a specifically disclosed conjugate or an unspecifically disclosed conjugate to treat the various disorders described, and the unspecifically disclosed conjugate can be converted into a specified conjugate in vivo after administration to the patient. Conventional methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985.

[0324] Furthermore, within the scope of the present invention, any element (especially when referred to with respect to the conjugates of the present invention) is intended to include all isotopes or isotopic mixtures of said element (naturally occurring or synthetically prepared) in its natural abundance or in its isotopically enriched form. For example, references to hydrogen include within its scope 1 H. 2 H(D) and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12C, 13 C and 14 C and 16 O and 18 O. The isotope may be radioactive or non-radioactive. The radiolabeled conjugate of the present invention may comprise a 3 H. 11 C. 18 F. 122 I. 123 I. 125 I. 131 I. 75 Br, 76 Br, 77 Br and 82 Preferably, the radioisotope is selected from 3 H. 11 C and 18 F.

[0325] Some conjugates of the present invention may exist as atropisomers. Atropisomers are stereoisomers resulting from hindered rotation around a single bond, where the steric strain barrier to rotation is high enough to allow separation of the conformers. It should be understood that all such conformers and mixtures thereof are encompassed within the scope of the present invention.

[0326] When the conjugates according to the present invention have at least one stereocenter, they may exist as enantiomers or diastereomers, respectively. It should be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0327] If the method for preparing the conjugate according to the present invention produces a mixture of stereoisomers, these isomers can be separated by conventional techniques such as preparative chromatography. The conjugate can be prepared in racemic form, or a single enantiomer can be prepared by enantiospecific synthesis or by splitting. For example, the conjugate can be split into their component enantiomers by standard techniques, such as by forming a salt with an optically active acid (such as (-)-di-p-toluoyl-D-tartaric acid and / or (+)-di-p-toluoyl-L-tartaric acid), followed by fractional crystallization and regeneration of the free base. The conjugate can also be split by forming diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the conjugate can be split using a chiral column via high performance liquid chromatography (HPLC) or SFC. In some cases, there may be rotational isomers of the conjugate, which can be observed by 1H NMR, resulting in complex multiple peaks and peak integration in the 1H NMR spectrum.

[0328] In any method for preparing the conjugates of the invention, it may be necessary and / or desirable to protect sensitive or reactive groups in any of the molecules involved. This can be achieved by conventional protecting groups, such as Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. Protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0329] How to use

[0330] The present invention relates to a method for preventing, treating or ameliorating a Y2 receptor-mediated syndrome and / or a GLP-1 receptor-mediated syndrome, disorder or disease in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a conjugate, compound or pharmaceutical composition of the present invention.

[0331] The present invention relates to a method for preventing, treating or ameliorating the onset of a disorder, disease or condition in a subject in need thereof, or ameliorating any one or more symptoms of the disorder, disease or condition, comprising administering to a subject in need thereof an effective amount of a conjugate, compound or pharmaceutical composition of the present invention.

[0332] According to specific embodiments, the disease, disorder or condition is selected from obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and / or eczema.

[0333] According to specific embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four or more of the following effects: (i) reduce or improve the severity of the disease, disorder or condition being treated or symptoms associated therewith; (ii) reduce the duration of the disease, disorder or condition being treated or symptoms associated therewith; (iii) prevent the development of the disease, disorder or condition being treated or symptoms associated therewith; (iv) cause the regression of the disease, disorder or condition being treated or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder or condition being treated or symptoms associated therewith; vi) prevent recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (ix) improve survival of subjects suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0334] The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder, or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0335] As used herein, the terms "treat," "treatment," and "therapy" are all intended to mean an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition that is not necessarily identifiable in a subject, but is capable of being identified in a subject. The terms "treatment" and "treatment" may also refer to causing regression, preventing development, or at least delaying the development of a disease, disorder, or condition. In a specific embodiment, "treatment," "treatment," and "therapy" refer to mitigation, prevention of development or onset, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to improved survival of a subject suffering from a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0336] In one embodiment, the invention relates to a method for preventing, treating or delaying the onset of obesity in a subject in need thereof, or ameliorating obesity, or any one or more symptoms of obesity, comprising administering to a subject in need thereof an effective amount of a conjugate, a compound or a pharmaceutical composition of the invention.

[0337] In one embodiment, the invention provides a method of reducing body weight in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a conjugate, compound or pharmaceutical composition of the invention.

[0338] In some embodiments, the subject's body weight is reduced by, e.g., about 0.01% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 2% to about 3%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50%, relative to the subject's body weight before administration of any of the conjugates, compounds, pharmaceutical compositions, forms, or medicaments of the invention described herein, or compared to a control subject not receiving any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein.

[0339] In some embodiments, the reduction in body weight lasts, for example, for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0340] The present invention provides a method for preventing, treating, delaying the onset of, or ameliorating a syndrome, disorder, or disease, or any one or more symptoms of the syndrome, disorder, or disease, in a subject in need thereof, wherein the syndrome, disorder, or disease is selected from obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema, the method comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.

[0341] As used herein, metabolic syndrome refers to a subject having any one or more of the following: high blood sugar (e.g., high fasting glucose), high blood pressure, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglyceride levels (e.g., high triglycerides), large waist circumference (i.e., waist circumference), increased fat in the abdominal area, insulin resistance, glucose intolerance, elevated C-reactive protein levels (i.e., a pro-inflammatory state), and increased plasma plasminogen activator inhibitor-1 and fibrinogen content (i.e., a pro-thrombotic state).

[0342] The present invention relates to a method of reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a conjugate, a compound or a pharmaceutical composition of the present invention. In some embodiments, the subject's food intake is reduced, e.g., by between about 0.01% to about 0.1%, between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 2% to about 3%, between about 5% to about 10%, between about 10% to about 15%, between about 15% to about 20%, between about 20% to about 25%, between about 25% to about 30%, between about 30% to about 35%, between about 35% to about 40%, between about 40% to about 45%, or between about 45% to about 50%, relative to the subject's food intake prior to administration of any of the conjugates, compounds, compositions, formats, medicaments or combinations of the invention as described herein, or compared to a control subject that did not receive any of the conjugates, compounds, compositions, formats, medicaments or combinations of the invention as described herein.

[0343] In some embodiments, the reduction in food intake lasts, for example, for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0344] The present invention provides a method of reducing glycated hemoglobin (A1C) in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. In some embodiments, the subject's A1C is reduced, for example, by about 0.001% to about 0.01%, between about 0.01% to about 0.1%, between about 0.1% to about 0.2%, between about 0.2% to about 0.3%, between about 0.4% to about 0.5%, between about 0.6% to about 0.7%, between about 0.8% to about 0.9%, between about 1.2% to about 2.3%, between about 1.4% to about 2.5%, between about 1.6% to about 2.7%, between about 1.8% to about 2.9%, between about 2.1% to about 2.3%, between about 1.2% to about 2.5%, between about 1.2% to about 2.3%, between about 1.4% to about 2.5%, between about 1.6% to about 2.7%, between about 1.2% to about 2.8%, between about 1.2% to about 2.9%, between about 1.4% to about 2.5%, between about 1.2% to about 2.3%, between about 1.4% to about 2.5%, between about 1.2% to about 2.5%, between about 1.4% to about 2.5%, between about 1. Between about 0.3% and about 0.4%, between about 0.4% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 1.5%, between about 1.5% and about 2%, between about 2% and about 2.5%, between about 2.5% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, or between about 9% and about 10%.

[0345] In other embodiments, a method for reducing fasting blood glucose levels in a subject in need thereof is provided, the method comprising administering an effective amount of a conjugate, compound, or pharmaceutical composition of the invention to a subject in need thereof. Prior to administration of any of the conjugates, compounds, compositions, forms, drugs, or combinations of the invention described herein, relative to the subject's fasting blood glucose levels, or compared to a control subject who has not received any of the conjugates, compounds, compositions, forms, drugs, or combinations of the invention described herein, the fasting blood glucose levels may be reduced to less than about 140 to about 150 mg / dL, less than about 140 to about 130 mg / dL, less than about 130 to about 120 mg / dL, less than about 120 to about 110 mg / dL, less than about 110 to about 100 mg / dL, less than about 100 to about 90 mg / dL, or less than about 90 to about 80 mg / dL.

[0346] The present invention relates to a method for regulating the Y2 receptor activity and GLP-1 receptor activity of a subject in need thereof, the method comprising administering an effective amount of a conjugate, compound or pharmaceutical composition of the present invention to a subject in need thereof. As used herein, "regulating" means increasing or decreasing receptor activity.

[0347] In some embodiments, an effective amount of a conjugate or compound of the invention, or a form, composition, or medicament thereof, is administered to a subject in need thereof once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, or eight times a day. In other embodiments, an effective amount of a conjugate or compound of the invention, or a form, composition, or medicament thereof, is administered to a subject in need thereof once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month, three times a month, or four times a month.

[0348] Another embodiment of the invention includes a method of preventing, treating, delaying the onset of a disease, disorder or syndrome in a subject in need thereof, or ameliorating a disease, disorder or syndrome, or one or more symptoms thereof, comprising administering a conjugate, compound or pharmaceutical composition of the invention to a subject in need thereof in combination therapy. In certain embodiments, the combination therapy is a second therapeutic agent. In certain embodiments, the combination therapy is surgical therapy.

[0349] As used herein, the term "in combination with" in the context of administering two or more therapies to a subject refers to the use of more than one therapy.

[0350] As used herein, combination therapy involves administering one or more additional therapeutic agents, or one or more surgical therapies, to a subject in need thereof simultaneously with an effective amount of a conjugate or compound of the invention, or a form, composition, or medicament thereof. In some embodiments, one or more additional therapeutic agents or surgical therapies may be administered on the same day as an effective amount of a conjugate of the invention, and in other embodiments, one or more additional therapeutic agents or surgical therapies may be administered together with an effective amount of a conjugate or compound of the invention in the same week or month.

[0351] The present invention also contemplates the use of combination therapy to prevent, treat, delay the onset of, or ameliorate any of the diseases, disorders, syndromes, or symptoms described herein in a subject in need thereof, the combination therapy comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention in combination with any one or more of the following therapeutic agents: dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonists (e.g., short-acting GLP-1 receptor agonists, such as exenatide and lixisenatide; intermediate-acting GLP-1 receptor agonists, such as liraglutide; long-acting GLP-1 receptor agonists, such as extended-release exenatide, albiglutide, dulaglutide); sodium-glucose co-converter SGLT-2 inhibitors (e.g., canaglifozin, dapaglifozin, empaglifozin); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., immediate-release bromocriptine); biguanides (e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpromazine, glimepiride, glipizide, glyburide, glibenclamide, gliprexa, gliprexa, glyburide, gliprexa, gliprexa, glyburide, glyburide, glyburide, tolsulfuronamide, tolbutamide, acetophenonesulfonylcyclohexylamide, amoxicillin, etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, daglitazone, englitazone, neglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, the dosage of one or more additional therapeutic agents is reduced when provided in combination with a conjugate or compound of the invention. In some embodiments, when used in combination with a conjugate or compound of the invention, the additional therapeutic agent may be used at a lower dosage than when each is used alone.

[0352] The present invention contemplates the use of combination therapy to prevent, treat, delay the onset of any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof, or ameliorate any of the diseases, disorders, syndromes or symptoms, comprising administering an effective amount of a conjugate, compound or pharmaceutical composition of the present invention to a subject in need thereof in combination with surgical therapy. In certain embodiments, the surgical therapy may be obesity surgery (e.g., gastric bypass surgery, such as biliojejunal gastric bypass surgery; sleeve gastrectomy; adjustable gastric banding surgery; biliopancreatic diversion duodenal switch; indwelling gastric balloon; gastric plication; and combinations thereof).

[0353] In embodiments where one or more additional therapeutic agents or surgical therapies are administered on the same day as an effective amount of a conjugate or compound of the invention, the conjugate or compound of the invention may be administered before, after, or simultaneously with the additional therapeutic agent or surgical therapy. The use of the term "in combination" does not limit the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) may be administered to a subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks ago), at the same time, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) a second therapy is administered to a subject.

[0354] Implementation

[0355] The present invention also provides the following non-limiting embodiments.

[0356] Embodiment 1 is a conjugate comprising a glucagon-like peptide (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide and a second linker peptide, wherein the first linker peptide is optionally absent.

[0357] Embodiment 2 is the conjugate according to embodiment 1, wherein the cyclic PYY peptide is represented by Formula I or a derivative or a pharmaceutically acceptable salt thereof:

[0358]

[0359] in

[0360] p is 0 or 1;

[0361] m is 0, 1, 2, 3, 4 or 5;

[0362] n is 1, 2, 3 or 4;

[0363] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0364] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0365] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0366] Z4 is K, A, E, S or R;

[0367] Z7 is A or K;

[0368] Z9 is G or K;

[0369] Z 11 is D or K;

[0370] Z 22 A or K;

[0371] Z 23 is S or K;

[0372] Z 26 A or H;

[0373] Z 30 is L, W or does not exist,

[0374] The premise is that only when q is 1, Z 30 does not exist;

[0375] Z 34 for

[0376] Z 35 for

[0377] The derivative is a compound of formula I modified by one or more methods selected from amidation, acylation and pegylation.

[0378] Embodiment 3 is the conjugate according to embodiment 2, wherein the cyclic PYY peptide is represented by formula I or a derivative or a pharmaceutically acceptable salt thereof, wherein:

[0379] p is 0 or 1;

[0380] m is 0, 1, 2, 3, 4 or 5;

[0381] n is 1, 2, 3 or 4;

[0382] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0383] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-,

[0384] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0385] Z4 is K, A, E, S or R;

[0386] Z7 is A or K, wherein the amino side chain of K is optionally substituted by

[0387] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0388] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0389] Z9 is G or K, wherein the amino side chain of K is optionally substituted by

[0390] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0391] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0392] Z 11 is D or K, wherein the amino side chain of K is optionally substituted by

[0393] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0394] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0395] Z 22 is A or K, wherein the amino side chain of K is optionally substituted by

[0396] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0397] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0398] Z 23 is S or K, wherein the amino side chain of K is optionally substituted by

[0399] wherein i is an integer from 0 to 24, and X=Br, I or Cl,

[0400] -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl;

[0401] Z 26 A or H;

[0402] Z 30 is L;

[0403] Z 34 for and

[0404] Z 35 for

[0405] wherein X is an electrophilic group, and the Br, Cl or I of the X electrophilic group is replaced in the conjugation reaction to form the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0406] Embodiment 4 is the conjugate according to embodiment 2, wherein the cyclic PYY peptide is represented by formula I or a derivative or a pharmaceutically acceptable salt thereof, wherein:

[0407] p is 0 or 1;

[0408] m is 0, 1, 2, 3 or 5;

[0409] n is 1, 2 or 4;

[0410] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q can be 1;

[0411] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-,

[0412] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0413] Z4 is K, A, E, S or R;

[0414] Z7 is A or K, wherein the amino side chain of K is substituted by

[0415] -C(O)CH2Br,

[0416] Z9 is G or K, wherein the amino side chain of K is substituted by

[0417] -C(O)CH2Br,

[0418] Z 11 is D or K, wherein the amino side chain of K is substituted by

[0419] -C(O)CH2Br,

[0420] Z 22 is A or K, wherein the amino side chain of K is substituted by

[0421] -C(O)CH2Br,

[0422] Z 23 is S or K, wherein the amino side chain of K is substituted by

[0423] -C(O)CH2Br,

[0424] Z 26 A or H;

[0425] Z 30 is L;

[0426] Z 34 for and

[0427] Z 35 for wherein the Br is replaced in the conjugation reaction to form the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0428] Embodiment 5 is the conjugate according to embodiment 1 or 2, wherein the cyclic PYY peptide is selected from SEQ ID NO: 1-54, or a pharmaceutically acceptable salt thereof.

[0429] Embodiment 6 is the conjugate according to embodiment 5, wherein the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

[0430] Embodiment 7 is the conjugate according to any one of embodiments 1 to 6, wherein the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide.

[0431] Embodiment 8 is the conjugate according to embodiment 7, wherein the cyclic PYY peptide comprises a chemical linker.

[0432] Embodiment 9 is a conjugate according to embodiment 8, wherein the chemical linker comprises a chemical linker selected from C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain having 2 to 24 PEG units, a linker comprising an acyl group, and an alkyl chain comprising 2 to 10 carbon atoms.

[0433] Embodiment 10 is a conjugate according to any one of embodiments 7 to 9, wherein Z7, Z9, Z 11 , Z 22 and Z 23Only one of the two is a lysine, and the lysine is covalently linked to a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide.

[0434] Embodiment 11 is the conjugate according to embodiment 10, wherein Z in Formula I 11 For lysine.

[0435] Embodiment 12 is a conjugate according to any one of embodiments 1 to 11, wherein the GLP-1 peptide comprises an amino acid sequence selected from SEQ ID NOs: 56-59.

[0436] Embodiment 13 is the conjugate according to embodiment 12, wherein the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO:57.

[0437] Embodiment 14 is a conjugate according to any one of embodiments 1 to 13, wherein the first linker peptide comprises an amino acid sequence selected from SEQ ID NOs: 60-83.

[0438] Embodiment 15 is a conjugate according to embodiment 14, wherein the first linker peptide comprises the amino acid sequence of SEQ ID NO:60.

[0439] Embodiment 16 is a conjugate according to any one of embodiments 1 to 15, wherein the hinge-Fc region peptide comprises an amino acid sequence selected from SEQ ID NOs: 84-90.

[0440] Embodiment 17 is a conjugate according to embodiment 16, wherein the hinge-Fc region peptide comprises the amino acid sequence of SEQ ID NO:84 or SEQ ID NO:85.

[0441] Embodiment 18 is a conjugate according to any one of embodiments 1 to 17, wherein the second linker peptide comprises an amino acid sequence selected from SEQ ID NOs: 93-112.

[0442] Embodiment 19 is a conjugate according to embodiment 18, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO:93, 94, 95, 106 or 111.

[0443] Embodiment 20 is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and wherein the cyclic PYY peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33 or 34.

[0444] Embodiment 21 is a conjugate according to embodiment 20, wherein the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO:113.

[0445] Embodiment 22 is a conjugate according to embodiment 20, wherein the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO:136.

[0446] Embodiment 23 is a conjugate according to embodiment 21 or 22, wherein a cysteine ​​residue between residues 287 and 289 of the GLP-1 fusion peptide is covalently linked to a lysine residue at residue 7, 9, 11, 22 or 23 of the cyclic PYY peptide.

[0447] Embodiment 24 is a conjugate according to embodiment 23, wherein the cysteine ​​residue is located at residue 288 of the GLP-1 fusion peptide.

[0448] Embodiment 25 is a conjugate according to any one of embodiments 20 to 24, wherein the lysine residue is located at residue 11 of the cyclic PYY peptide.

[0449] Embodiment 26 is a conjugate according to any one of embodiments 20 to 25, wherein the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide via a chemical linker on the cyclic PYY peptide.

[0450] Embodiment 27 is a conjugate according to embodiment 26, wherein the chemical linker is selected from C(O)CH2, polyethylene glycol (PEG)8-triazolyl-CH2CH2CO-PEG4, a PEG chain having 2 to 24 PEG units, a linker comprising an acyl group, or an alkyl chain comprising 2 to 10 carbon atoms.

[0451] Embodiment 28 is a conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the conjugate comprises a sequence selected from SEQ ID NOs: 225-262, or a pharmaceutically acceptable salt thereof.

[0452] Embodiment 29 is a conjugate according to any one of embodiments 1 to 28, wherein the GLP-1 fusion peptide is a monomer.

[0453] Embodiment 30 is a conjugate according to any one of embodiments 1 to 28, wherein the GLP-1 fusion peptide is a dimer.

[0454] Embodiment 31 is a method for preparing the conjugate according to any one of embodiments 1 to 30, comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced onto the side chain of the cyclic PYY peptide, preferably the amino side chain of a lysine residue of the cyclic PYY peptide, with the sulfhydryl group of the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, thereby forming a covalent bond between the cyclic PYY peptide and the GLP-1 fusion peptide.

[0455] Embodiment 32 is a method according to embodiment 31, wherein the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, thereby reacting the reduced cysteine ​​residue with the electrophile.

[0456] Embodiment 33 is a method according to embodiment 32, wherein the azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

[0457] Embodiment 34 is a pharmaceutical composition comprising the conjugate according to any one of embodiments 1 to 30 and a pharmaceutically acceptable carrier.

[0458] Embodiment 35 is a method for treating or preventing obesity in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to embodiment 34 to the subject in need thereof.

[0459] Embodiment 36 is a method according to embodiment 35, wherein administering an effective amount of the pharmaceutical composition to the subject in need thereof results in a reduction in body weight of about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 25%, compared to the subject's body weight prior to administration of the pharmaceutical composition.

[0460] Embodiment 37 is a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and / or eczema, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 34.

[0461] Embodiment 38 is a method according to embodiment 37, wherein the disease or disorder is obesity.

[0462] Embodiment 39 is a method according to embodiment 37, wherein the disease or disorder is type 1 diabetes.

[0463] Embodiment 40 is a method according to embodiment 37, wherein the disease or disorder is type II diabetes.

[0464] Embodiment 41 is a method according to embodiment 37, wherein the disease or disorder is metabolic syndrome.

[0465] Embodiment 42 is a method according to embodiment 37, wherein the disease or disorder is kidney disease.

[0466] Embodiment 43 is a method according to embodiment 37, wherein the disease or disorder is non-alcoholic steatohepatitis (NASH).

[0467] Embodiment 44 is a method according to embodiment 37, wherein the disease or disorder is non-alcoholic fatty liver disease (NAFLD).

[0468] Embodiment 45 is a method of reducing at least one of food intake or body weight in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 34.

[0469] Embodiment 46 is a method according to embodiment 45, wherein administering an effective amount of the pharmaceutical composition to the subject in need thereof results in a decrease in food intake of about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% compared to the subject's food intake prior to administration of the pharmaceutical composition.

[0470] Embodiment 47 is a method of modulating Y2 receptor activity and GLP-1 receptor activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to Embodiment 34.

[0471] Embodiment 48 is a method according to any one of embodiments 35 to 47, wherein the pharmaceutical composition is administered via injection.

[0472] Embodiment 49 is a method according to embodiment 48, wherein the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0473] Embodiment 50 is a method according to any one of embodiments 35 to 49, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0474] Embodiment 51 is a method according to any one of embodiments 35 to 50, wherein the pharmaceutical composition is administered daily, weekly or monthly to the subject in need thereof.

[0475] Embodiment 52 is a method according to embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times or six times a day.

[0476] Embodiment 53 is a method according to embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times or six times per week.

[0477] Embodiment 54 is a method according to embodiment 51, wherein the pharmaceutical composition is administered once, twice, three times or four times per month.

[0478] Embodiment 55 is a kit comprising the conjugate according to any one of embodiments 1 to 30 or the pharmaceutical composition according to embodiment 34, preferably the kit further comprises an injection device.

[0479] Embodiment 56 is a method for preparing a pharmaceutical composition comprising the conjugate according to any one of embodiments 1 to 30, the method comprising combining the conjugate with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0480] Embodiment 57 is an isolated hinge-Fc region platform peptide, wherein the hinge-Fc region platform peptide comprises: a hinge-Fc region peptide, wherein the hinge-Fc region peptide preferably comprises an amino acid sequence selected from SEQ ID NO: 84-90; a first linker peptide, wherein the first linker peptide preferably comprises an amino acid sequence selected from SEQ ID NO: 60-83, and the first linker peptide is optionally deleted; and a second linker peptide, wherein the second linker peptide comprises an amino acid sequence selected from SEQ ID NO: 91-112, wherein the first linker peptide is connected to the amino terminus of the hinge-Fc region peptide, and the second linker peptide is connected to the carboxyl terminus of the hinge-Fc region peptide.

[0481] Embodiment 58 is an isolated hinge-Fc region platform peptide according to embodiment 57, wherein the hinge-Fc region peptide comprises the amino acid sequence of SEQ ID NO:84.

[0482] Embodiment 59 is an isolated hinge-Fc region platform peptide according to embodiment 57 or 58, wherein the first linker peptide comprises the amino acid sequence of SEQ ID NO:60.

[0483] Embodiment 60 is an isolated hinge-Fc region platform peptide according to any one of embodiments 57 to 59, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106 or 111.

[0484] Embodiment 61 is a conjugate comprising the hinge-Fc region platform peptide covalently linked to a target peptide at the second linker peptide.

[0485] Embodiment 62 is a conjugate according to embodiment 61, wherein the target peptide is a cyclic PYY peptide represented by Formula I or a derivative or pharmaceutically acceptable salt thereof:

[0486]

[0487] in

[0488] p is 0 or 1;

[0489] m is 0, 1, 2, 3, 4 or 5;

[0490] n is 1, 2, 3 or 4;

[0491] q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1;

[0492] BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-,

[0493] -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-;

[0494] Z4 is K, A, E, S or R;

[0495] Z7 is A or K;

[0496] Z9 is G or K;

[0497] Z 11 is D or K;

[0498] Z 22 A or K;

[0499] Z 23 is S or K;

[0500] Z 26 A or H;

[0501] Z 30 is L, W or does not exist,

[0502] The premise is that only when q is 1, Z 30 does not exist;

[0503] Z 34 for

[0504] Z 35 for The derivative is a compound of formula I modified by one or more methods selected from amidation, acylation and pegylation.

[0505] Embodiment 63 is a method for preparing a conjugate of the hinge-Fc region platform peptide and a target peptide, the method comprising forming a covalent bond between the second linker peptide and the target peptide.

[0506] Example

[0507] synthesis

[0508] The compounds or conjugates of the present invention can be synthesized according to general synthetic methods known to those skilled in the art. The following description of the synthesis is for illustrative purposes and is in no way intended to be a limitation of the present invention.

[0509] The NTSC cyclic PYY (NTSC-PYY) analogs or derivatives of the present invention can be synthesized by a variety of conventional methods known for forming continuous peptide bonds between amino acids, and preferably by solid phase peptide synthesis (SPPS) as generally described in Merrifield (J. Am. Chem. Soc., 85: 2149-2154 (1963)), using an automatic peptide synthesizer, traditional bench-scale synthesis, or a combination of these two methods. Conventional methods for peptide synthesis involve condensation between the free amino group of one amino acid residue whose other reactive functional groups have been appropriately protected and the free carbonyl group of another amino acid whose reactive functional groups have also been appropriately protected. Examples of condensing agents commonly used for peptide bond formation include diisopropylcarbodiimide (DIC) with or without 1-hydroxybenzotriazole (HOBT) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HBTU), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HATU), 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HCTU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyOxim), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), bromo-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyBroP), etc.

[0510] Automated peptide synthesis methods can be performed at room temperature (rt) or at elevated temperature, preferably by applying microwave heating, as described by Yu (J. Org. Chem., 57:4781-4784 (1992)) and as recently improved by Palasek (J. Pept. Sci., 13:143-148 (2007)).

[0511] The compounds of the present invention (C-terminal amides) can be conveniently prepared using the N-α-FMOC (9-fluorenylmethoxycarbonyl) protected amino acid method, whereby an appropriately protected N-α-FMOC protected amino acid is coupled to a conventional solid phase resin using a suitable coupling agent. Suitable conventional commercially available solid phase resins include: Rink amide MBHA resin, Rink amide AM resin, Tentagel S RAM resin, FMOC-PAL-PEGPS resin, SpheriTide Rink amide resin, ChemMatrixRink resin, Sieber amide resin, TG Sieber resin, and the like. The resin-bound FMOC amino acid can then be deprotected by exposure to a 20% piperidine solution in N,N dimethylformamide (DMF) or 1-methyl-2-pyrrolidone (NMP), which treatment is used to selectively remove the FMOC protecting group. The additional FMOC protected amino acid is then coupled and deprotected in sequence to produce the desired resin-bound protected peptide. In some cases, it may be necessary to use an orthogonally reactive protecting group for another amine in the peptide sequence that will withstand the FMOC deprotection conditions. Protecting groups such as 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt), both of which can be removed by treatment with 1% trifluoroacetic acid (TFA / dichloromethane (DCM), or preferably allyloxycarbonyl (alloc; removed by treatment with Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium(0)) / PhSiH3 (phenylsilane)), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde; removed by treatment with 2-3% hydrazine / DMF), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde; removed by treatment with 2-3% hydrazine / DMF) can be effectively used in this case.

[0512] In conventional peptide synthesis methods, the reactive side chains of alpha amino acids are usually protected with suitable protecting groups throughout the synthesis to render them inert to coupling and deprotection protocols. Although a number of protecting groups for amino acid side chains are known in the art, the following protecting groups are most preferred herein: tert-butyl (t-Bu) for serine, threonine, glutamic acid, aspartic acid, and tyrosine; trityl (Trt) for asparagine, glutamine, cysteine, homocysteine, and histidine; tert-butyloxycarbonyl (Boc) for the epsilon-amino groups of tryptophan and lysine; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine. These protecting groups are removed upon treatment with strong acids, such as high concentrations of trifluoroacetic acid (TFA).

[0513] At the completion of the SPPS, the resin-bound, side-chain protected peptide is deprotected and concomitantly cleaved from the resin using a cleavage mixture consisting primarily of (TFA) together with various combinations of carbocation scavengers such as triisopropylsilane (TIPS), water, phenol and anisole. The crude solid peptide is then isolated by precipitation of the peptide / mixture filtrate with cold ether. In the special case of a Sieber resin-bound protected peptide, cleavage of the protected peptide from the resin can advantageously be achieved with repeated treatment with 1-2% TFA in DCM without causing side chain deprotection. Once isolated, further manipulation of the protected peptide can be performed in a solution phase reaction. Finally, the protected peptide is globally deprotected using a separate treatment with a cleavage mixture and precipitated as described above. The crude peptide thus obtained is then dissolved in a primarily aqueous solvent system containing an organic co-solvent such as acetonitrile or ethanol at low concentrations (approximately <4 mg / mL). Upon raising the pH of the solution to >5, the peptide then undergoes an intramolecular cyclization reaction to form the corresponding crude NTSC PYY analog of the present invention. The NTSC PYY analogs thus formed can be purified using purification techniques generally known in the art. The preferred peptide purification method used herein is reverse phase high performance liquid chromatography (HPLC). The purified peptides are then characterized by liquid chromatography / mass spectrometry (LC / MS).

[0514] General protocols and methods for preparing the cyclic PYY peptides of the present invention are described in U.S. Patent Application No. 15 / 794,231 filed on October 26, 2017 and U.S. Patent Application No. 15 / 794,171 filed on October 26, 2017. The contents of both applications are hereby incorporated by reference in their entirety.

[0515] It should be understood that the following examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereto will be suggestive to those skilled in the art, and will be included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0516] Example 1: Expression of Glucagon-like Peptide 1 (GLP-1) Fusion Peptides from Transiently Transfected Mammalian Cells Dahe Purification

[0517] DNA constructs of GLP-1 fusion peptides were generated by cloning the synthesized gene fragments into pCDNA3.1 derived mammalian expression vectors using different designed endonucleases and restriction enzymes. The synthesized gene fragments were designed to contain a DNA sequence encoding one or all of the following components of the fusion protein (from N-terminus to C-terminus): a GLP-1 peptide or a GLP-1 variant peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide containing a Cys for conjugation to a cyclic PYY peptide.

[0518] GLP-1 fusion protein in ExpiCHO-S TM Expression was performed by transiently transfecting cells with purified plasmid DNA of the fusion protein expression construct in ExpiCHO-S cells (ThermoFisher Scientific; Waltham, MA, catalog number A29127) according to the manufacturer's recommendations. Briefly, the ExpiCHO-S cells were cultured in a shaking incubator set at 37°C, 8% CO2, and 125 RPM. TM Cells in ExpiCHO TM The cells were kept suspended in expression medium (ThermoFisherScientific, catalog number A29100). The cells were passaged and diluted to 6.0×10 6 cells / ml, thus maintaining cell viability at 98% or higher. TM Transient transfection was performed using the CHO Transfection Kit (ThermoFisher Scientific Catalog No. A29131). For each ml of diluted cells to be transfected, one milligram of plasmid DNA was used and diluted to OptiPRO TM In SFM complex medium. ExpiFectamine was used at a 1:3 ratio (v / v, DNA:reagent) TM CHO reagent and also diluted into OptiPRO TM The diluted DNA and transfection reagent were combined for one minute to allow DNA / lipid complexes to form and then added to the cells. After overnight incubation, the ExpiCHO TM Feed and ExpiFectamine TM The CHO enhancer was added to the cells. The cells were cultured at 37°C with shaking for five days, after which the culture supernatant was harvested.

[0519] Transiently transfected ExpiCHO-S were harvested by clarification via centrifugation (30 min, 6000 rpm) followed by filtration (0.2 μPES membrane, Corning; Corning, NY). TMThe culture supernatant of cell.Use Pall Centramate Tangential Flow Filtration system (Pall Centramate Tangential Flow Filtration system) first large-scale transfection (5 to 20 liters) is concentrated 10 times.10xDPBS, pH7.2 is added to the supernatant to reach 1X final concentration, then AKTAFPLC chromatography system is used, and is loaded into balanced (DPBS, pH7.2) HiTrapMabSelect Sure protein A column (GE Healthcare; Little Chalfont, United Kingdom) at a relative concentration of about 20mg protein per milliliter of resin.After loading, sequentially use 10 times of column volumes of DPBS, pH7.2 washing column.In some cases, include 20 times of column volumes of 100mMTris, 2.5M NaCl, 50mM sodium octanoate (pH 9) and 10 times of column volumes of DPBS (pH 7.2) additional washing.With 10 times of column volumes of 0.1M sodium acetate (Na) pH3 elution protein. The protein fraction was immediately neutralized by eluting at 20% of the elution fraction volume into a tube containing 2.0 M Tris, pH 7. Peak fractions were pooled and the pH was adjusted to about 7 with additional Tris as needed. The purified protein was filtered (0.2 μ) and eluted on a BioTek Synergy TM The concentration was determined by absorbance at 280 nm on a HTM spectrophotometer. TM HPLC system) to assess the quality of the purified protein. The turbidimetric LAL assay ( -T, Associates of Cape Cod; Falmouth, MA) to measure endotoxin levels.

[0520] Alternatively, GLP-1 fusion proteins were expressed in Expi293F cells (ThermoFisher Scientific, catalog number A14527) by transiently transfecting cells with purified plasmid DNA of the fusion protein expression construct according to the manufacturer's recommendations. Briefly, Expi293F cells were cultured in an Expi293 incubator set at 37°C, 8% CO2, and 125 RPM. TM The cells were kept suspended in expression medium (ThermoFisher Scientific, catalog number A1435101). The cells were passaged and diluted to 2.5×10 6 cells / ml, thus maintaining cell viability at 95% or higher. TMTransient transfection was performed using the OptiMEM 293 transfection kit (Thermo Fisher Scientific catalog number A14525). For each ml of diluted cells to be transfected, one milligram of plasmid DNA was used and diluted into OptiMEM TM ExpiFectamine was used at a ratio of 1:2.6 (v / v, DNA:reagent) in SFM complex medium. TM 293 reagent, dilute the reagent into OptiMEM TM 20 minutes, allowing the DNA / lipid complex to form, and then added to the cells. TM Feed and ExpiFectamine TM 293 enhancer was added to the cells. The cells were cultured at 37°C with shaking for four days, after which the culture supernatant was harvested.

[0521] Clarify by centrifugation (30min, 6000rpm), filter (0.2μPES membrane, Corning) afterwards, harvest the culture supernatant of transiently transfected Expi293F cells.10x DPBS, pH7.2 is added to the supernatant to reach 1X final concentration, then use AKTAFPLC chromatography system, load to balanced (DPBS, pH7.2) HiTrap MabSelect Sure protein A column (GE Healthcare) with the relative concentration of about 20mg protein / ml resin.After loading, wash the column with 10 times of column volumes of DPBS pH 7.2. Elute protein with 10 times of column volumes of 0.1M sodium acetate pH 3. Neutralize protein fraction immediately by eluting into a tube containing 2.0M Tris, pH 7 with 20% elution fraction volume. Peak fractions are merged and pH is adjusted to about 5.5 with other Tris when needed. Filter and purify protein (0.2μ), and in BioTek Synergy TM The concentration was determined by absorbance at 280 nm on a HTM spectrophotometer. TM HPLC system) to assess the quality of the purified protein. The turbidimetric LAL assay ( -T, Associates of Cape Cod) to measure endotoxin levels.

[0522] A GLP-1 fusion protein having a first linker peptide comprising the amino acid sequence of SEQ ID NO: 61, wherein the first linker peptide is a polyglycine linker, is expressed at low levels and has a low purification yield. Therefore, when engineering a GLP-1 fusion protein, it is disadvantageous to use polyglycine for the first linker peptide in this experiment.

[0523] The GLP-1 fusion protein with the second linker peptide comprising the amino acid sequence of SEQ ID NO: 100 was truncated and determined to be not of the expected molecular weight when analyzed by mass spectrometry. Therefore, the use of the second linker peptide of SEQ ID NO: 100 in this experiment is not advantageous when engineering a GLP-1 fusion protein.

[0524] Additionally, when preparing the GLP-1 fusion peptide construct, the carboxyl-terminal lysine of the hinge Fc region peptide (ie, human IgG4) was deleted from the final construct to avoid potential proteolytic activity.

[0525] Example 2: Preparation of GLP-1 fusion peptide coupled cyclic PYY peptide conjugate

[0526] Reduction of GLP-1 fusion protein

[0527] Method A

[0528] The purified GLP-1 fusion protein is a mixture of a recombinant protein and an engineered second linker peptide, which contains at least one cysteine ​​residue in each linker, which is intramolecularly disulfide-bonded to another cysteine ​​or bonded to an external cysteine ​​residue in the cytosol or growth medium. To such a heterologous GLP-1 fusion protein solution (5-12 mg / mL TrisOAc solution, pH 5.4-6.4), an excess of phosphine reducing agent 1,3,5-triaza-7-phosphadamantane (PTA, 15-40 equivalents) is added, followed by 100 mM EDTA (final concentration 1 mM-2 mM). The resulting reaction mixture is gently stirred at room temperature until the target cysteine ​​residue is completely reduced (4h-16h). The released cysteine ​​and the remaining PTA are removed by desalting chromatography.

[0529] Method B

[0530] An excess of the phosphine reducing agent tris(2-carboxyethyl)phosphine (TCEP, 4 eq-25 eq) was added to a solution of heterologous GLP-1 fusion protein (5-15 mg / mL in TrisOAc, pH 5.0-6.5), followed by 100 mM EDTA (final concentration 1 mM). The resulting reaction mixture was gently stirred overnight at room temperature and then applied to a desalting column equilibrated with TrisOAc (pH 7.0). The fully reduced GLP-1 fusion protein (hinge disulfide bond cleavage) was treated with dehydroascorbic acid (DHAA, 10 eq) for 2 hours, and the reduced GLP-1 fusion peptide was then oxidized to obtain a complete GLP-1 fusion peptide with an engineered cysteine ​​on a completely uncapped second linker peptide.

[0531] Preparation of GLP-1 fusion peptide coupled to cyclic PYY peptide conjugate

[0532] A TrisOAc solution (3 mg / mL-10 mg / mL, pH 5.5) of the reduced GLP-1 fusion peptide was added to a deionized water solution of the cyclic PYY peptide (2.6 to 3.0 equivalents, 10 mg / mL-15 mg / mL). In order to protect the reactive thiol from metal-catalyzed oxidation, a 100 mM EDTA solution was added to the reaction mixture until the concentration of EDTA was 1 mM. The pH of the reaction solution was raised to pH 8.0-8.2 by dropwise addition of Tris buffer (1 M, pH 9.1). The reaction was carried out at room temperature for 3-7 hours and then at 5 ° C for 18 hours. LCMS showed that the conjugation was complete and that the GLP-1 fusion cyclic PYY peptide conjugate was formed at greater than 95%. The reaction was directly subjected to protein A purification to remove excess cyclic PYY peptide, or quenched by adding 5 equivalents of cysteine ​​(to cap the remaining reactive cyclic PYY peptide). The resulting crude conjugate was purified by hydrophobic interaction chromatography (HIC) and then immediately adsorbed on protein A. The final product was obtained by elution of the conjugate (sodium acetate, pH 3.5) and subsequent neutralization with Tris buffer to pH 7-7.5. Alternatively, if HIC purification has been performed before, the HIC-purified conjugate can be exchanged to the desired buffer by a PD-10 column without further protein A polishing.

[0533] The conjugation reaction can also be performed at low temperature (5°C) and monitored by LCMS (approximately 48 hours).

[0534] In some cases, the active cysteine ​​in the second linker peptide of the GLP-1 fusion protein is blocked by bromoacetamide to form a stable thioether bond. These acetamide-modified GLP-1 fusion proteins are prepared as follows: an excess of phosphine reducing agent 1,3,5-triaza-7-phosphadamantane (PTA, 15 equivalents-20 equivalents) is added to the GLP-1 fusion protein solution (5mg / mL-12mg / mL TrisOAc solution, pH5.4-6.4), followed by 100mM EDTA (final concentration 1mM-2mM). The resulting reaction mixture was gently stirred at room temperature for 18 hours. 15 equivalents-20 equivalents of bromoacetamide were added to the GLP-1 fusion peptide solution, and the pH of the reaction mixture was adjusted to pH7.5-8.1 with Tris buffer (1M, pH 9.1). After 2 hours, the reaction mixture was purified by hydrophobic interaction chromatography (HIC), followed by protein A fine purification.

[0535] The isolated yields of selected GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates are provided in Table 8. It was observed that when the first linker peptide was an AP linker (as in GLP-1 fusion peptide 64 (GF64)) and the second linker peptide was a G4A linker (as in GLP-1 fusion peptide 8 (GF8)), the isolated yield of the GLP-1-coupled cyclic PYY peptide conjugate was poor.

[0536] Table 8: Isolated yields of selected GLP-1 coupled cyclic PYY peptide conjugates

[0537]

[0538]

[0539] Example 3: Characterization of GLP-1 fusion peptide coupled cyclic PYY peptide conjugate

[0540] The analytical characterization of the GLP-1 fusion peptide coupled cyclic PYY peptide conjugate was performed using (i) hydrophobic interaction chromatography (HIC), (iii) intact mass measurement by LC-ESIMS, (iii) size exclusion chromatography (SEC). The results of the analytical characterization of the GLP-1 fusion peptide coupled cyclic PYY peptide conjugate and the conjugation method are shown in Table 9.

[0541] Table 9: Analytical data of GLP-1 coupled cyclic PYY peptide conjugates .

[0542]

[0543]

[0544] Example 4: In vitro assay

[0545] Conjugates were screened for functional activity and in vitro potency in a cell-based assay that measured intracellular cAMP using the Lance competitive cAMP immunoassay (Perkin Elmer, Waltham, Massachusetts) according to the kit instructions. Clonal HEK293 cells stably expressing mouse or human GLP-1R or NPY2 receptor (Y2R) were used in the assay. Cells expressing GLP1R were thawed and suspended in HBSS, 5mM HEPES, 0.1% BSA, 0.5mM IBMX. Cells were mixed with the kit anti-cAMP antibody and added to serial dilutions of the conjugates in HBSS, 5mM HEPES, 0.1% BSA in a 384-well white opti-plate microplate. After incubation for 10 minutes at room temperature, the immunoassay detection mixture was added. The assay plate was read as a TR-FRET assay on an Envision plate reader (excitation 320nm, emission 615nm and 665nm), and the data were used to calculate compound EC using Prism statistical software (GraphPad Software San Diego). 50 value.

[0546] To determine the activity of compounds at the Y2R receptor, HEK293 cells expressing Y2R were cultured in DMEM-high glucose medium (Cellgro) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 1% penicillin / streptomycin and 600 μg / ml G418, and plated overnight in 384-well plates in medium without G418. On the day of the assay, the cell growth medium was removed from the cells and 6 μl of conjugate (2X) in 5 mM HEPES, 500 uM IBMX in HBSS and 0.1% BSA were added. Next, 6 μl of stimulation buffer containing forskolin (2X, 5 uM final concentration) and LANCE cAMP antibody (1:100) was added to the cells. After incubation at room temperature for 25 minutes, 12 μl of detection mixture was added and cAMP concentration was quantified in the LANCE cAMP immunoassay.

[0547] Data analysis

[0548] Data from the Envision plate reader are expressed as relative fluorescence units (RFU), calculated as (615 nm / 665 nm) x 10,000. All samples were measured in triplicate. The unknown cAMP concentration in each well was interpolated from a reference standard of known cAMP concentration included in each plate. 50 、Log(EC 50), HillSlope(nH), Top and Bottom parameters were derived by plotting cAMP concentration values ​​on the logarithmic compound concentrations fitted to the 4-P model using a nonlinear weighted least squares application.

[0549] Table 10: In vitro data of cyclic PYY conjugates coupled to GLP-1 fusion peptides .

[0550]

[0551]

[0552] Table 11: GLP-1R and NPYR2 in vitro potency of GLP-1 fusion peptides and GLP-1 fusion cyclic PYY conjugates

[0553]

[0554]

[0555] Comparison of the GLP-1R potency of SEQ ID NOs 135, 134, 146 and 176 with SEQ ID NOs 238, 242, 262, 253 and 241 showed that the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO: 58) lost GLP-1R potency after conjugation with the PYY peptide. Based on these results, the use of the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO: 58) was not advantageous in this experiment.

[0556] Human NPY2R primary screening assay : The method used for in vitro screening of NPY2R potency of GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates is a cell-based assay designed to measure inhibition of forskolin-induced cAMP production by adenylate cyclase through modulation of the human NPY2RG protein-coupled receptor. NPY2R was activated in a dose-dependent manner by PYY analogs and controls, and forskolin-induced cAMP production was reduced in human NPY2R-transfected CHO-K1 cells (DiscoverX) as measured in a FRET-based competitive cAMP immunoassay.

[0557] Remove the frozen cells and thaw them in a 37°C water bath, add them to 40 mL of 1x DPBS, pH 7.2 (Gibco), and filter through a cell strainer. Centrifuge the cells at 450xg for 5 minutes and aspirate the supernatant. 6The density of cells / ml was resuspended in DMEM / high glucose, 10% HIFBS, 1% penicillin / streptomycin, 1% L-glutamine, 1% sodium pyruvate. The cells were distributed to a collagen-coated white 384-well plate at 40 μL / well to reach a final 5000 cells / well and incubated at 37°C, 5% CO2 for 16 to 24 hours. The cells in the assay plate were washed twice by adding 80 μL / well 1xDPBS and decanting the supernatant. Dilutions of samples and controls were prepared in 1x HBSS, 5mM HEPES, 0.002mM forskolin, 0.1% BSA, 0.5mM 3-isobutyl-1-methylxanthine (IBMX), and each sample of 20 μL / well was added to the designated well and incubated at room temperature for 30 minutes. Then 20 μL / well cAMP detection reagent mixture was added to each assay plate and incubated at room temperature for 2 to 24 hours. Plates were read on a plate reader. All samples were measured in quadruplicate. Data were analyzed by plotting raw LANCE cAMP values ​​over log compound concentrations fitted to a 4-P model using a nonlinear weighted least squares application in the R environment.

[0558] Human GLP-1R Primary Screening Assay : The method used for in vitro screening of GLP-1 fusion proteins and GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates for GLP-1R potency is a cell-based assay designed to measure cAMP production through modulation of the human GLP-1R Gs-protein coupled receptor. Following concentration-dependent activation of GLP-1R by GLP-1 fusion peptides and GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates, cAMP accumulation in human GLP-1R transfected HEK cells was measured in a TRFRET-based competitive cAMP assay.

[0559] On the day of the assay, human GLP-1R transfected HEK cells were thawed and plated at 0.5 × 10 6 Cells / ml were resuspended in 1XHBSS, 5mM HEPES, 0.1% BSA and 1mM IBMX, and 10 μl was added to each well of a 384-well plate (5000 cells / well). The samples were diluted in 1X HBSS, 5mM HEPES, 0.1% BSA, added to 10 μL / well of the assay plate, and incubated at room temperature for 30 minutes. The cAMP detection reagent was added at 20 μL / well. The plate was incubated at room temperature for 2 hours and then read on a plate reader. All samples were measured four times. The data were analyzed by plotting the original LANCE cAMP values ​​on the logarithmic compound concentrations fitted with a 4-P model using a nonlinear weighted least squares application in the R environment.

[0560] Example 5: Xylose analysis of GLP-1 fusion peptide and GLP-1 fusion peptide coupled cyclic PYY conjugate

[0561] Sample preparation : To prepare samples for peptide mapping, each protein molecule (1 mg / ml): GLP-1 fusion peptide coupled cyclic PYY conjugate (SEQ ID NO: 244), GF40 (SEQ ID NO: 152) and GF34 (SEQ ID NO: 146) was diluted 1:4 in 8M guanidine / HCl buffered at pH 8.0. An aliquot of 1M DTT (Sigma 43816-10ML, BioUltra) was added to these samples to reach a final concentration of 25mM; the samples were then incubated at 37°C for 1 hour. Alkylation reactions were performed using iodoacetamide (Sigma, A3221-10VL) or N-methylmaleimide (NEM) (Sigma, E3876). Freshly prepared 1M alkylating agent was added to reach approximately 50mM, and the samples were incubated at room temperature in the dark for 60 minutes. The alkylation reaction is then quenched by adding 15 μl of 1M DTT to every 400 μl reaction mixture (Sigma 43816-10ML, BioUltra). Using Zeba spin desalting column (Thermo, catalog number 89833), according to the manufacturer's protocol, in 50mM Tris, 1mM CaCl2, pH=8.0, the sample is desalted. After adding 1 μl of 1 μg / μL trypsin reconstructed in 50mM acetic acid (provided with lyophilized trypsin), the sample is digested for 4 hours at 37°C with trypsin (Promega sequencing grade, V511A). Trypsin digestion is quenched by adding 0.6 μl of 100% TFA to the digested protein of 60 μL aliquots. The sample is resuspended in a micro vial and placed in an automatic sampler set to 4°C for LC / MS analysis.

[0562] Liquid chromatography and mass spectrometry:Use Agilent Infinity 1290UHPLC (Agilent Technologies, part number: G1330B, G4226A, G4220A, G4212A), inject 2 μg (about 10 μl) of digested protein into AgilentAdvanceBio Peptide Map Micro Bore Rapid Resolution Column (1×150mm, 2.7 μm, part number: 863600-911) at a flow rate of 0.1 mL / min. The column temperature was maintained at 65°C. Mass spectrometry grade HPLC solvents (0.1% formic acid, and B: 0.1% formic acid solution of 100% ACN) were purchased from VWR (part number: LC 452-1, LC 441-1). A 50-minute gradient of 0.1% formic acid solution of 2% to 40% ACN was used to elute proteolytic peptides from the column. The column effluent was introduced into an Orbitrap Q-Exactive mass spectrometer (Thermofisher Scientific) via a heated electrospray ionization probe (HESI) using a positive spray voltage of 3.5 kV, a sheath gas of 20 (arbitrary units), an auxiliary gas of 7 (arbitrary units), an ion transfer tube of 299° C., and a vaporizer of 100° C. Data-dependent acquisition was performed by sequentially dissociating the top 5 most abundant peptide ions observed in the full MS scan. Mass analysis was performed using a precursor scan set to Orbitrap detection, 70,000 resolution, a mass range of 150-2000 m / z, an automatic gain control (AGC) target value of 1.0e6, and a maximum injection time of 50 milliseconds. One microscan spectrum was collected in distribution mode. The precursor decision criteria were monoisotopic precursor selection peptides, charge states: 2-7, dynamic exclusion: 6.0 seconds, and a precursor intensity threshold of 5e4. The precursor peptides were separated by a quadrupole, and a separation window of 1.6 m / z was sent to the collision cell. The normalized collision energy result of 28 (arbitrary units) was used for high energy collision dissociation (HCD) of peptide ions. Product ions were transferred to the orbital region for mass analysis with the orbital region settings of 17,500 resolution, 200-2000 m / z range, AGC target value of 5e5, maximum injection time of 100 ms, and 1 microscan of spectra acquired in centroid mode.

[0563] Data analysisDatabase searches were performed on raw LC–MS / MS data using Byonic software (version 2.15.7) (Protein Metrics). The following parameters were used for data searches: precursor ion mass tolerance, 8 ppm; product ion mass tolerance, 20 ppm for HCD spectra; variable modifications included cysteine ​​carbamidomethylation, or N-methylmaleimide (NEM), cysteine ​​DTT adduct formation, cysteine ​​peptide YY (PYY) conjugates when present, deamidation of asparagine, serine xylose, and serine and threonine O-glycosylation of 78 mammalian O-linked glycans from the Byonic glycan library. For all searches, a full trypsin-specific search with a maximum of two missed cleavages was selected. The protein false discovery rate (FDR) was set to 1%. The protein database contains the amino acid sequence of each protein. All Byonic search results were processed in Bytional software (version 2.15.296) (ProteinMetrics), with a minimum Byonic score of 15, a maximum alt run score / primary rank score of 0.99, a maximum precursor m / z error of 8ppm, and a XIC area window of 2 minutes to filter peptides. The serine xylosylation level was reported by considering all peptide XIC peak areas of the isoforms constituting the joint region. The xylosylation at each serine residue was then calculated as the sum score of the total XIC peak area. The Andromeda search engine in MaxQuant software version 1.6.1 (MaxPlankInstitute) was also used to perform database searches to obtain PTM site location probabilities. The mass of the PYY structure corresponding to the fully cyclized PYY and trypsin cleavage was obtained using BIOVIA drawing software. The data are reported in Table 12.

[0564] Table 12: Xylose modification

[0565]

[0566] Xylose modification in linker serine: site identification and modification levels

[0567] ND = not detected by MS / MS; NQ = not quantified; Na = linker serine position not applicable to the molecule; **SEQ ID NO: parentheses indicate position number of SEQ ID NO: 152.

[0568] Table 12 summarizes site-specific xylosylation identified and quantified using peak integration in Byological, where the XIC% based on the peak / peaks of interest (putative modification, i.e., serine xylosylation) was normalized to the sum of all species contributing to site-specific xylosylation according to the following formula:

[0569] Modified XIC% = (XIC of modified peptide / ∑XIC of peptide counterpart) × 100

[0570] All three molecules studied consist of the same first linker peptide AS(G4S)2 linking the GLP-1 peptide and the hinge-Fc region peptide. However, due to differences in the GLP-1 fusion peptide (GF40) present in SEQ ID NO: 152 and the GLP-1 fusion peptide (GF34) present in both SEQ ID NO: 244 and 146, the positions of three serine residues in SEQ ID NO: 152 differ from SEQ ID NO: 244 and 146, as shown in Table 12. Analysis of the peptides confirmed three site-specific serine residue xylosylation events at Ser-41, Ser-46, Ser-51 in SEQ ID NO: 244 and 146 and one serine residue xylosylation event at Ser-33 in SEQ ID NO: 152. Quantification of site-specific xylosylation is only possible to a limited extent, as most of the xylosylated peptide species co-elute in reversed phase liquid chromatography. For example, in SEQ ID NO:244, Ser-41 is about 9%, while Ser-46 and Ser-51 are about 7%. Similarly, all three sites of SEQ ID NO:146 are xylosylated at about 5%. It was found that xylosylation of SEQ ID NO:152 occurs to a lesser extent, at about 0.1%.

[0571] The G4S linker region of therapeutic proteins has been shown to be susceptible to xylosylation, with the level of xylosylation varying based on linker length (Wen et al., "The propensity for xylosylationin (G4S) n linkersoccurs whenn>2," Anal.Chem.85:4805-12 (2013); Sphar et al., Protein Sci.22:1739-53 (2013), Sphar et al., mAbs 6:904-14 (2014)). The first linker peptide AS(G4S)2 linker present in all three molecules showed xylosylation at all or some serine residues, and based on these data, the use of a serine-containing first linker peptide was not favorable in this experiment when preparing GLP-1 fusion peptide constructs.

[0572] Example 6: In vitro human plasma stability of GLP-1 fusion peptides

[0573] On the day of establishing the stability sample, fresh whole blood containing sodium heparin anticoagulant was received from 4 normal human donors. The plasma of fresh whole blood was processed by centrifugation at 3500RPM for 15 minutes, and then the plasma was separated. The plasma from all four donors was combined, filtered through a 0.2μ filter, and warmed to 37°C. 20nM of GLP-1 fusion peptide and GLP-1 fusion peptide-coupled cyclic PYY conjugate dual agonist (DA) analogs were added to the sample, and the zero time sample was immediately collected and stored at -80°C until analysis. The samples were incubated at 37°C and collected at the specified time points, and stored at -80°C until analysis. On the day of analysis, the samples at zero time and subsequent time points were thawed and analyzed together in a functional cell-based bioassay.

[0574] The method used to detect and quantify the levels of bioactive GLP-1 fusion peptide and GLP-1 fusion peptide-conjugated cyclic PYY conjugate DA analogs in human plasma samples is a cell-based bioassay designed to measure cAMP production through modulation of the human GLP-1R G-protein coupled receptor. Following concentration-dependent activation of GLP-1R by GLP-1 fusion protein and GLP-1 fusion peptide-conjugated cyclic PYY peptide conjugate, cAMP accumulation in human GLP-1R transfected HEK cells was measured in a TRFRET-based competitive cAMP assay. Human GLP-1R transfected HEK cells were thawed on the day of the assay and plated at 0.5 × 10 6 Cells / ml were resuspended in 1X HBSS, 5mM HEPES, 0.1% BSA and 1mM IBMX, and 10 μl was added to each well of a 384-well plate (5000 cells / well). The stability samples were thawed and diluted to 20% plasma in an assay buffer consisting of 1X HBSS, 5mM HEPES, 0.1% BSA, 5mM EDTA, protease inhibitors, and subsequent dilutions were made with 20% normal human plasma in the assay buffer. Reference standards of known concentrations of each compound were prepared in an assay buffer containing 20% ​​normal human plasma. Standards and samples were added to each assay plate at 10 μL / well and incubated at room temperature for 30 minutes. cAMP detection reagent was added at 20 μL / well. The plates were incubated at room temperature for 2 hours and then read on a plate reader.

[0575] All samples were measured in quadruplicate. Compound reference standard curves were generated using log-transformed concentrations, nonlinear fit, log vs. response (variable slope), and EC values ​​were determined for upper and lower limits of quantitation. 10 and EC 90The concentration of the stability samples of each compound was extrapolated from the corresponding reference standard curve. The remaining percentage of biological activity of each stability sample was determined relative to the corresponding zero time sample and reported as the initial plasma concentration% during the 168 hour period. Initial plasma concentration% = [average stability sample] / [average zero time] × 100.

[0576] result : GLP-1 fusion peptides SEQ ID NO: 144 (GF32), 148 (GF36), 145 (GF33), 151 (GF39), 146 (GF34), 147 (GF35), 149 (GF37), 152 (GF40) and dulaglutide control were incubated in vitro in human plasma at 37°C for 7 days, and functional stability was measured in an in vivo GLP-1R cAMP functional assay ( Figure 1A ). The GLP-1 fusion protein with the GLP-1 peptide SEQ ID NO:58 (exendin 4 (1-39)) showed the greatest stability, followed by SEQ ID NO:57 ((A8G, G22E, R36G) GLP-1 (7-37)) and the dulaglutide control, with SEQ ID NO:56 ((A8S, A30E) GLP-1 (7-36)) being the least stable. Based on these results, the use of an Fc fusion protein with the GLP-1 peptide SEQ ID NO:56 was not advantageous in this experiment.

[0577] GLP-1 fusion peptides SEQ ID NO: 152 (GF40) and 146 (GF34) and their corresponding GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates SEQ ID NO: 248 and 262, and another GLP-1 fusion peptide SEQ ID NO: 153 (GF41) were incubated in vitro in human plasma for 7 days at 37°C along with a dulaglutide control. Functional stability was measured in an in vitro GLP-1R cAMP functional assay ( Figure 1B ). The stability of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugates was comparable to the corresponding unconjugated fusion proteins, indicating that conjugation of the PYY peptide did not affect the stability of the GLP-1 peptide. The GLP-1 fusion peptide SEQ ID NO: 146 (GF34) and the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate SEQ ID NO: 262 had the greatest stability, and SEQ ID NOs: 152 (GF40), 248, and 153 (GF41) had similar stability to the dulaglutide control.

[0578] Example 7: In vivo mouse stability assay

[0579] method

[0580] LCMS Method: Plasma samples were processed by immunoaffinity capture using an anti-human Fc antibody, followed by trypsin digestion and reverse phase LC-MS / MS analysis on a triple quadrupole mass spectrometer. Peptides at the N-terminus of GLP-1, namely HGE (HGEGTFTSDVSSYLEEQAAK (SEQ ID NO: 263) for GLPD30 and HGEGTFTSDLSK (SEQ ID NO: 264) for GLPD31, were monitored as surrogates for molecules containing active GLP1. Peptides located on the Fc, namely VVS (VVSVLTVLHQDWLNGK (SEQ ID NO: 265), were monitored as surrogates for total Fc levels. Peptide YYA (YYASLR (SEQ ID NO: 266)), located on the cyclic PYY peptide, was monitored as a surrogate for PYY levels. Calibration standard curves and quality control samples were prepared by spiking plasma with reference standards and processed simultaneously using the same procedure as test samples.

[0581] result

[0582] The plasma exposure level of the N-terminal of the intact GLP-1 fusion peptide portion was measured in the LCMS assay after 72 hours of administration in mice and reported as the percentage of the HGE N-terminal GLP-1 fusion peptide portion sequence level relative to the Fc level (HGE% of Fc). The results in Table 13 demonstrate that the first linker peptides of SEQ ID NOs: 62 and 66 containing AP repeat sequences are not as stable as the first linker peptide of SEQ ID NO: 60 containing G4A repeat sequences. Based on these results, the use of GLP-1 fusion peptides with a first linker peptide containing AP repeat sequences is not advantageous in this experiment.

[0583] Table 13: In vivo stability of GLP-1 fusion peptide and GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate in mice

[0584]

[0585] BQL = Below Quantitation Limit

[0586] Acute and subchronic pharmacodynamic studies in DIO mice

[0587] All rodents used in these studies were maintained in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Janssen R&D, Spring House, PA. Animals were housed under standard temperature and humidity conditions with a 12-hour light / 12-hour dark cycle and free access to food and water (unless otherwise stated). Twenty-week-old male DIO (60% kcal% high-fat diet for 15 weeks) C57BL / 6T mice (TaconicLaboratory) were used. Animals were randomly divided based on body weight. For intraperitoneal (IP) glucose tolerance test (IPGTT), mice were administered vehicle, dulaglutide (Eli Lilly; purchased from Myoderm, Norristown, PA) or compound 1-4 (SEQ ID NO: 113, SEQ ID NO: 225, SEQ ID NO: 136, SEQ ID NO: 229, respectively) (n = 8 / group). Twenty-four hours later (after overnight fasting), mice were administered dextrose (1 g / kg) IP. Blood glucose was measured at designated times using a One Touch Ultra glucometer (LifeScan), and plasma insulin was measured at 0 and 10 minutes (Meso Scale Discovery). For acute food intake (FI) and weight loss (WL) studies, vehicle, dulaglutide or compound were administered to singly housed DIO mice, and food intake and body weight were measured for 3 days (n=8 / group). Data are shown in FIG. 2A to FIG. 2B , FIG. 3A to FIG. 3B and FIG. 4A to FIG. 4B middle. FIG. 2A to FIG. 2B , FIG. 3A to FIG. 3B and FIG. 4A to FIG. 4B The data in show that the GLP-1 fusion peptide has the desired GLP-1R efficacy and has enhanced efficacy when conjugated to the cyclic PYY peptide SEQ ID NO: 27. FIG. 5A to FIG. 5B and FIG. 6A to FIG. 6B The data in , establish that the upper hinge of the GLP-1 fusion peptide contributes to GLP-1R potency.

[0588] Example 8: In vivo study in cynomolgus monkeys

[0589] method

[0590] Efficacy studies

[0591] Monkeys were selected and randomized for compound treatment based on baseline (average of 3 days prior to day 0) food intake (primary-weighted 80%) and body weight (secondary-weighted 20%) measured during the run-in and vehicle treatment periods. The study lasted for 9 weeks, 2 weeks of acclimation / training, 3 weeks of vehicle treatment every 3 days (Q3D) and baseline food intake measurements, 12 days of treatment (5 doses, Q3D), and 2 weeks of washout. Animals were dosed 7 times with vehicle at approximately 8 AM and 5 times Q3D with one of the test articles (at a dosing volume of 0.5 mL / kg). The doses for each group were as follows: dulaglutide (0.0125 mg / kg, n=11), compound 4 (high dose; 0.0148 mg / kg, n=11), compound 4 (low dose; 0.0074 mg / kg, n=6). Daily caloric intake was measured, and body weight was measured Q3D.

[0592] Exposure response analysis

[0593] The concentrations of compound 4 and dulaglutide in plasma samples in vivo in cynomolgus monkeys were measured using two analytical methods.

[0594] Ligand Binding Assay (LBA) Exposure Analysis of Compound 4 and Dulaglutide in Cynomolgus Monkeys was performed by an adaptable electrochemiluminescent immunoassay (ECLIA) method using a MesoScale Discovery (MSD) Sector Imager S600 (Meso Scale Diagnostics, Rockville, M.D. USA). The form of active GLP-1 measured for both Compound 4 and Dulaglutide was described as follows: Analytes were captured with GLP-1 N-terminal (7-17) specific mAb (biotin-anti-GLP1 (7-37, 7-36, amide, free NT) mAb) and detected with anti-human Fc specific mAb (SulfoTag-R10). A 5-parameter logistic fit with 1 / F2 standard curve weighting was used and the results were analyzed in Watson LIMS. TM Raw data regression was performed using Fischer(R) software (Thermo Fischer Scientific, Waltham, MA USA).

[0595] LC-MS / MS analysis of compound 4 and dulaglutide in cynomolgus monkeys was also performed by a bottom-up tryptic assay for quantification of individual parts of the molecule. For the tryptic assay, analytes were purified from plasma samples using immunoaffinity capture with anti-human Fc antibodies, followed by tryptic digestion and analysis on a SCIEX Triple Quad TMReverse phase LC-MS / MS analysis was performed on a 5500 LC-MS / MS system (CIEX, Concord, Ontario, Canada). Human IgG4 (Sigma, catalog number MSQC7) labeled with stable isotopes [13C6,15N4-arginine] and [13C6,15N2-lysine] was used as an internal standard and added at the beginning of the immunoaffinity capture step. The N-terminal peptide of GLP-1, HGE (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 263), was monitored as a surrogate for molecules containing active GLP-1, as the HGE peptide is essential for its activity.

[0596] result

[0597] The dual agonist according to the embodiment of the present application comprises a GLP-1 fusion (GF) peptide covalently linked to a cyclic PYY peptide. The dual agonist is composed of a recombinant glucagon-like peptide 1 (GLP-1) peptide or a GLP-1 variant peptide fused to a hinge-Fc region via a first linker peptide (N-terminal linker peptide) and a cyclic PYY peptide chemically conjugated to a target cysteine ​​contained in a second linker peptide (C-terminal linker peptide) of the recombinant GLP-1 fusion (GF) peptide. Each component of the dual agonist is empirically selected based on several key parameters, including but not limited to potency, stability, post-translational modification, conjugation preparation yield, and biophysical properties for each receptor.

[0598] GLP-1 peptide variant SEQ ID NO: 57 was selected as the GLP-1 peptide variant. GLP-1 peptide-1 variant SEQ ID NO: 56 had lower potency in vivo and lower stability in in vitro human plasma stability studies ( Figure 1A), and therefore is disadvantaged in this experiment. The GLP-1 peptide variant SEQ ID NO:58 unexpectedly loses GLP-1R potency when conjugated to the cyclic PYY peptide (Table 11), see, for example, the GLP-1R potency of SEQ ID Nos:135, 134, 146 and 176 with SEQ ID NOs:238, 242, 262, 253 and 241, indicating that the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO:58) loses or has significantly reduced GLP-1R potency after conjugation to some PYY peptides. Based on these results, the use of the GLP-1 fusion peptide with exendin 4 (1-39) (SEQ ID NO:58) is disadvantageous in this experiment. The GLP-1 peptide variant SEQ ID NO:58 additionally contains a NG deamidation potential motif. When glycine is the first amino acid in the first linker peptide, SEQ ID NO:59 forms a NG deamidation potential motif at the GLP-1 peptide / first linker peptide junction. In addition, GF28 (SEQ ID NO:140) comprising the GLP-1 peptide SEQ ID NO:59 had less potency than dulaglutide in both food intake (FI) and % change in body weight (BW) (0.1% and 0.2%, respectively). Fig. 10A and Fig. 10B ).

[0599] SEQ ID NO:60 was selected as the first linker peptide (N-terminal linker peptide). The GLP-1 fusion protein comprising the first peptide linker SEQ ID NO:61 had low expression and purification yields and was not conducive to producing the desired amount of the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate in this experiment. When the second terminal peptide was a G4A repeat sequence, the first linker peptide comprising an AP repeat sequence had a poor yield (see, e.g., Example 2 and Table 8) and was less stable in vivo compared to SEQ ID NO:60 (see, e.g., Table 13). Based on these results, the use of an AP repeat linker was not favorable in this experiment. Shorter linkers resulted in reduced potency (see, e.g., SEQ ID NOs:152, 153, 154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 201 FIG. 10A to FIG. 10B and Fig.12 140 in ). Thus, based on these results, the use of shorter linkers was disadvantaged in this experiment. The use of serine-containing linkers was disadvantaged in this experiment based on the propensity of serine residues to be xylosylated (see, e.g., Table 12).

[0600] The GLP-1 fusion peptide GF19 (SEQ ID NO: 131) comprises the GLP-1 variant peptide SEQ ID NO: 57, the first linker peptide SEQ ID NO: 60 and the hinge-Fc region peptide SEQ ID NO: 84, and the fusion peptide exhibited comparable efficacy to dulaglutide in both food intake (FI) and % change in body weight (BW) (see, e.g., Fig. 6A and Figure 6B ).

[0601] The GLP-1 fusion peptides GF1 (SEQ ID NO: 113, compound 1), GF19 (SEQ ID NO: 131), and GF24 (SEQ ID NO: 136, compound 3) all showed comparable efficacy to dulaglutide in terms of food intake and % change in body weight (see e.g. FIG. 2A to FIG. 2B , FIG. 6A to FIG. 6B and FIG. 3A to FIG. 3B GF1 (SEQ ID NO: 113, compound 1) and GF24 (SEQ ID NO: 136, compound 3) also showed comparable efficacy to dulaglutide in terms of glucose tolerance ( FIG. 4A to FIG. 4B and FIG. 7A to FIG. 7B ).

[0602] However, when the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate SEQ ID NO:251 was prepared by conjugating the cyclic PYY peptide (SEQ ID NO:27) to the second linker peptide SEQ ID NO:92 of GF19 (SEQ ID NO:131), there was no additional pharmacological effect on food intake or % body weight change compared to dulaglutide (9.3% and 1.2%, respectively). Fig.11A and Fig. 11B ). Additional second linker peptides were tested but were disadvantaged in this experiment for at least the following reasons: (a) SEQ ID NO:95 had a low production yield when the first linker peptide was an AP repeat sequence, (b) SEQ ID NO:100 was truncated during recombinant expression, (c) the hinge Fc region peptide without the second linker peptide contained the target cysteine ​​at the C-terminus of the hinge Fc region peptide for conjugation to the PYY peptide, which was truncated during protein expression, indicating that a second linker peptide was required to produce an intact protein suitable for PYY peptide conjugation, (d) the serine-containing second linker peptide had the potential for xylosylation, and (e) shorter second linker peptides conjugated less efficiently at lower purity.

[0603] While the GLP-1 fusion peptide coupled cyclic PYY peptide conjugate SEQ ID NO: 225 (Compound 2) with a second linker peptide SEQ ID NO: 94 showed significantly greater potency than dulaglutide in both FI and % change in BW (see, e.g., Figure 2Aand Figure 2B ), but surprisingly had significant protein loss during storage at 4°C for 2 weeks in a high concentration liquid formulation. The comparable GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate SEQ ID NO: 229 with a second linker peptide SEQ ID NO: 93 was stable and maintained protein levels in a high concentration liquid formulation during storage at 4°C and 40°C for two weeks (Table 14). The second linker peptide was the only difference between the two molecules, so SEQ ID NO: 93 was selected as the second linker peptide in this experiment.

[0604] Table 14: Protein stability in high concentration liquid formulations during storage for two weeks at 4°C and 40°C

[0605]

[0606] Several modifications are required for the functional activity of the cyclic PYY peptide to confer the required in vivo stability for an agent with an extended half-life and to enable attachment to a GLP-1 fusion peptide while maintaining potency. Synthetic peptide chemistry is employed to prepare cyclic PYY peptides containing these modifications, and to chemically conjugate to a GLP-1 fusion peptide to generate a GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate.

[0607] A cyclic PYY peptide was chosen for conjugation to a GLP-1 fusion peptide to explore and compare modifications of PYY peptide potency and stability. The PYY peptide was cyclized to stabilize multiple proteolytic sites along the peptide backbone.

[0608] Cyclic PYY peptides were selected for conjugation to GLP-1 fusion peptides to explore the spacing and size of the N-terminal to C-terminal bonds of the cyclic PYY peptides and the effects on potency and stability. These cyclic PYY peptides contain variations in the number of carbons in the loop and alter the positions of cysteine ​​and homocysteine ​​for selective attachment of the N-terminus to the C-terminus. See, e.g.

[0609] a) βA2 linked to hCys31 SEQ ID NO:25

[0610] b) βA2 linked to Cys30 SEQ ID NO: 28 and 29

[0611] c) G2 is linked to Cys30 SEQ ID NO: 27 and 30

[0612] d) γAba2 linked to hCys31 SEQ ID NO:34

[0613] Comparison of the ability of the amidated C-terminal tyrosine at position 36 in the stabilized cyclic PYY peptides with N-Me arginine substituted at position 35 (N-MeR35) (SEQ ID Nos: 27, 28, 33, and 34) and a reduced amide bond between positions 35 and 36 (psi35,36) (SEQ ID NOs: 24, 25, 29, and 30) and maintaining potency (see, e.g., U.S. Patent Publication No. 2018 / 0117170, which is incorporated herein by reference in its entirety). In some cases, the N-Me35 modification resulted in reduced potency compared to the psi35,36 modification (see, e.g., U.S. Patent Publication No. 2018 / 0117170), Table 3 shows that SEQ ID NO: 102 with psi35,36 modification is 12 times more potent at the human Y2 receptor than SEQ ID NO: 122 with N-Me35 modification.

[0614] Unexpectedly, the N-MeR35 modification in the GLP-1 fusion peptide-coupled cyclic PYY peptide conjugate SEQ ID 229 comprising cyclic PYY peptide SEQ ID 27 had equal or greater potency than the psi35,36 modification in SEQ ID NO: 232 comprising cyclic PYY peptide SEQ ID NO: 30 (Table 11).

[0615] In this experiment, the cyclic PYY peptide SEQ ID NO: 27 was selected as the PYY peptide based on potency and stability and the combination of determined components resulting in lower commodity costs and convenience of reagent sources. The natural amino acid glycine attached to the N-terminus of the cysteine ​​at position 30 near the C-terminus has potency and stability comparable to the highest level of potency and stability achieved by any cyclic PYY peptide tested when the cyclic PYY peptide was conjugated to the GLP-1 fusion peptide. Because the cyclic PYY peptide SEQ ID NO: 27 has sufficient potency and stability, the cysteine ​​at position 30 was selected relative to the homocysteine ​​at position 31. Therefore, more expensive and less available non-natural amino acids were not required in this experiment. N-MeR35, which is used to stabilize the C-terminal amidated tyrosine at position 36, is a cheaper and more readily available reagent than the expensive psi35,36 modification. This eliminates the need for custom components and provides the same stability and potency.

[0616] In addition, it was determined that the efficacy does not require a PEG spacer, and it was also unexpectedly determined that the in vivo PYY stability does not require a PEG spacer. This is unexpected because it was determined that the mAb cyclic PYY peptide conjugate with a PEG spacer has increased in vivo stability (see, e.g., U.S. Patent Publication 2018 / 0117170, Table 4, which shows the remaining percentage of the amount of the complete compound relative to the total human mAb level in the blood sample from the mouse collected 48 hours after administration. Compound 1 containing a 12xPEG spacer has 90.8% remaining at 48 hours; Compound 2 containing a 6xPEG spacer has 65.6% remaining at 48 hours; and Compound 3 without a PEG spacer has only 51% remaining at 48 hours). This result eliminates the need for a PEG component that would increase the cost of commodity manufacturing.

[0617] In this experiment, SEQ ID NO: 229, which comprises the GLP-1 fusion peptide SEQ ID NO: 136 to which two stable cyclic PYY peptides SEQ ID NO: 27 were added, was selected as the cyclic PYY peptide conjugate coupled to the GLP-1 fusion peptide. SEQ ID NO: 229 exhibited additional pharmacodynamic efficacy from Y2R conjugation in both FI and BW relative to SEQ ID NO: 136 (with only GLP1R conjugation) ( FIG. 3A to FIG. 3B ), while showing GLP1R potency on glucose tolerance comparable to dulaglutide and SEQ ID NO: 136 ( FIG. 4A to FIG. 4B and FIG. 7A to FIG. 7B ).

[0618] The effects of dulaglutide and SEQ ID NO: 229 (Compound 4) on FI and BW were compared in DIO mice. Based on the comprehensive exposure-response nonlinear regression analysis of the available studies (6 studies for Compound 4 and 7 studies for dulaglutide; doses 0.03 nmol / kg to 1.0 nmol / kg), at doses that resulted in clinically relevant active GLP-1 (HGE) exposures on day 3 (single subcutaneous dose 0.3 nmol / kg), Compound 4 showed additive pharmacology relative to dulaglutide in reducing FI and BW at matched active GLP1 (HGE) exposures through engagement of the conjugate's cyclic PYY peptide via the Y2R ( FIG. 8A to FIG. 8B Based on the exposure-response nonlinear regression of compound 4 (SEQ ID NO: 229), at the matched 0.3 nmol / kg dulaglutide HGE concentration (single SC dose), the calculated FI and % change in BW were approximately 2.1-fold and 1.5-fold that of dulaglutide, respectively.

[0619] In overweight cynomolgus monkeys, exposure-response analyses based on individual PK and caloric intake ( FIG. 9A to FIG. 9B) showed that at clinically relevant dulaglutide exposures (1–2 nM (including C at steady state) following QW SC dosing of 1.5 mg in patients with T2DM), trough and C max ) [Geiser et al., Clin Pharmacokinet 55(5):(2016)]), compound 4 resulted in approximately 1.7- to 2-fold (95% confidence interval: 1.3 to 3.3, PK, based on ligand binding assay [LBA], compared to dulaglutide at matched active GLP-1 exposures. Fig.9A ) or about 1.5 to 1.6 times (95% confidence interval: 1.0 to 2.8, PK, based on LC-MS / MSHE, Fig. 9B ) of the control group, indicating that the cyclic PYY peptide of Compound 4 has additive pharmacology.

[0620] It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. It will therefore be appreciated that the present invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention, as defined in this specification.

[0621] All documents cited herein are incorporated by reference.

[0622] Exemplary GLP-1 fused cyclic PYY peptide conjugates of the invention include:

[0623] SEQ ID NO:225

[0624] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0625] structure:

[0626]

[0627] SEQ ID NO:226

[0628] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0629] structure:

[0630]

[0631] SEQ ID NO:227

[0632] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0633] structure:

[0634]

[0635] SEQ ID NO:228

[0636] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0637] structure:

[0638]

[0639] SEQ ID NO:229

[0640] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0641] structure:

[0642]

[0643] SEQ ID NO:230

[0644] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0645] structure:

[0646]

[0647] SEQ ID NO:231

[0648] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0649] structure:

[0650]

[0651] SEQ ID NO:232

[0652] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0653] structure:

[0654]

[0655] SEQ ID NO:233

[0656] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0657] structure:

[0658]

[0659] SEQ ID NO:234

[0660] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0661] structure:

[0662]

[0663] SEQ ID NO:235

[0664] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0665] structure:

[0666]

[0667] SEQ ID NO:236

[0668] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0669] structure:

[0670]

[0671] SEQ ID NO:237

[0672] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0673] structure:

[0674]

[0675] SEQ ID NO:238

[0676] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0677] structure:

[0678]

[0679] SEQ ID NO:239

[0680] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0681] structure:

[0682]

[0683] SEQ ID NO:240

[0684] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0685] structure:

[0686]

[0687] SEQ ID NO:241

[0688] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0689] structure:

[0690]

[0691] SEQ ID NO:242

[0692] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0693] structure:

[0694]

[0695] SEQ ID NO:243

[0696] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0697] structure:

[0698]

[0699] SEQ ID NO:244

[0700] Name: GLP-1 fusion-[cyclo-(G2-COCH2-hC31), K(PEG24Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0701] structure:

[0702]

[0703] SEQ ID NO:245

[0704] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0705] structure:

[0706]

[0707] SEQ ID NO:246

[0708] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0709] structure:

[0710]

[0711] SEQ ID NO:247

[0712] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0713] structure:

[0714]

[0715] SEQ ID NO:248

[0716] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0717] structure:

[0718]

[0719] SEQ ID NO:249

[0720] Name: GLP-1 fusion-[cyclo-(G2-E30), S4, K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0721] structure:

[0722]

[0723] SEQ ID NO:250

[0724] Name: GLP-1 fusion-[cyclo-(G2-E30), S4, K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0725] structure:

[0726]

[0727] SEQ ID NO:251

[0728] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0729] structure:

[0730]

[0731] SEQ ID NO:252

[0732] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0733] structure:

[0734]

[0735] SEQ ID NO:253

[0736] Name: GLP-1 fusion-[cyclo-(gAba2-COCH2-hC30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0737] structure:

[0738]

[0739] SEQ ID NO:254

[0740] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0741] structure:

[0742]

[0743] SEQ ID NO:255

[0744] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0745] structure:

[0746]

[0747] SEQ ID NO:256

[0748] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0749] structure:

[0750]

[0751] SEQ ID NO:257

[0752] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0753] structure:

[0754]

[0755] SEQ ID NO:258

[0756] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0757] structure:

[0758]

[0759] SEQ ID NO:259

[0760] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, psi-(R35, Y36)]-PYY2-36 homodimer conjugate

[0761] structure:

[0762]

[0763] SEQ ID NO:260

[0764] Name: GLP-1 fusion-[cyclo-(βA2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0765] structure:

[0766]

[0767] SEQ ID NO:261

[0768] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 monomer conjugate

[0769] structure:

[0770]

[0771] SEQ ID NO:262

[0772] Name: GLP-1 fusion-[cyclo-(G2-COCH2-C30), K(Ac)11, N-Me-R35]-PYY2-36 homodimer conjugate

[0773] structure:

[0774]

Claims

1. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises a GLP-1 peptide, a first linker peptide, a hinge-Fc region peptide, and a second linker peptide, wherein the first linker peptide is optionally deleted, and wherein the cyclic PYY peptide is represented by Formula I or a derivative or a pharmaceutically acceptable salt thereof: in p is 0 or 1; m is 0, 1, 2, 3, 4 or 5; n is 1, 2, 3 or 4; q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1; BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-; Z4 is K, A, E, S or R; Z7 is A or K; Z9 is G or K; Z 11 is D or K; Z 22 A or K; Z 23 is S or K; Z 26 A or H; Z 30 is L, W or does not exist, The premise is that only when q is 1, Z 30 does not exist; Z 34 for Z 35 for The derivative is a compound of formula I modified by one or more methods selected from amidation, acylation and pegylation.

2. The conjugate according to claim 1, wherein the cyclic PYY peptide is represented by formula I or a derivative or a pharmaceutically acceptable salt thereof, wherein: p is 0 or 1; m is 0, 1, 2, 3, 4 or 5; n is 1, 2, 3 or 4; q is 0 or 1; the premise is that only when Z 30 When it does not exist, q is 1; BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -SCH2C(O)NH2-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-; Z4 is K, A, E, S or R; Z7 is A or K, wherein the amino side chain of K is optionally substituted by wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl; Z9 is G or K, wherein the amino side chain of K is optionally substituted by wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl; Z 11 is D or K, wherein the amino side chain of K is optionally substituted by wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl; Z 22 is A or K, wherein the amino side chain of K is optionally substituted by wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl; Z 23 is S or K, wherein the amino side chain of K is optionally substituted by wherein i is an integer from 0 to 24, and X = Br, I or Cl, -C(O)CH2Br, -C(O)CH2I or -C(O)CH2Cl; Z 26 A or H; Z 30 is L; Z 34 for and Z 35 for 3. The conjugate according to claim 1, wherein the cyclic PYY peptide is represented by Formula I or a derivative or a pharmaceutically acceptable salt thereof, wherein: p is 0 or 1; m is 0, 1, 2, 3 or 5; n is 1, 2 or 4; q is 0 or 1; the premise is that only when Z 30 When it does not exist, q can be 1; BRIDGE is -Ph-CH2-S-, -triazolyl-, -NHC(O)CH2S-, -(OCH2CH2)2NHC(O)CH2S, -NHC(O)- or -CH2S-; Z4 is K, A, E, S or R; Z7 is A or K, wherein the amino side chain of K is substituted by -C(O)CH2Br, Z9 is G or K, wherein the amino side chain of K is substituted by -C(O)CH2Br, Z 11 is D or K, wherein the amino side chain of K is substituted by -C(O)CH2Br, Z 22 is A or K, wherein the amino side chain of K is substituted by -C(O)CH2Br, Z 23 is S or K, wherein the amino side chain of K is substituted by -C(O)CH2Br, Z 26 is A or H, Z 30 is L; Z 34 for Z 35 for 4. The conjugate according to claim 1, wherein the cyclic PYY peptide is selected from SEQ ID NO: 1-54, or a pharmaceutically acceptable salt thereof.

5. The conjugate of claim 4, wherein the cyclic PYY peptide is selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

6. The conjugate according to any one of claims 1 to 5, wherein the GLP-1 fusion peptide is covalently linked to the cyclic PYY peptide at a lysine residue of the cyclic PYY peptide.

7. The conjugate according to claim 6, wherein Z7, Z9, Z 11 , Z 22 and Z 23 Only one of the two is a lysine, and the lysine is covalently linked to a cysteine ​​residue in the second linker peptide of the GLP-1 fusion peptide.

8. The conjugate according to claim 7, wherein Z in Formula I 11 For lysine.

9. The conjugate according to any one of claims 1 to 8, wherein the GLP-1 peptide comprises an amino acid sequence selected from SEQ ID NOs: 56-59.

10. The conjugate according to any one of claims 1 to 9, wherein the first linker peptide is present and comprises an amino acid sequence selected from SEQ ID NOs: 60-83.

11. The conjugate according to any one of claims 1 to 10, wherein the hinge-Fc region peptide comprises an amino acid sequence selected from SEQ ID NOs: 84-90.

12. The conjugate according to any one of claims 1 to 11, wherein the second linker peptide comprises an amino acid sequence selected from SEQ ID NO: 93-112.

13. The conjugate according to any one of claims 1 to 11, wherein the second linker peptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, 106 or 111.

14. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-224 and 267-274, and wherein the cyclic PYY peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

15. The conjugate according to claim 14, wherein a cysteine ​​residue between amino acid residues 287 to 289, preferably cysteine ​​residue 288, of the GLP-1 fusion peptide is covalently linked to a lysine residue at residue 7, 9, 11, 22 or 23 of the cyclic PYY peptide, preferably lysine residue 11 of the cyclic PYY peptide via a chemical linker.

16. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 231-238, 241-245, 247-250, 252-257 and 260-262, or a pharmaceutically acceptable salt thereof.

17. A method for preparing the conjugate according to any one of claims 1 to 16, the method comprising reacting an electrophile, preferably bromoacetamide or maleimide, introduced onto the side chain of the cyclic PYY peptide, preferably the side chain of a lysine residue of the cyclic PYY peptide, with a sulfhydryl group of a cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide, thereby forming a covalent bond between the cyclic PYY peptide and the GLP-1 fusion peptide.

18. The method of claim 17, wherein the cysteine ​​residue of the second linker peptide of the GLP-1 fusion peptide is reduced by contacting the GLP-1 fusion peptide with an excess of an azaphosphine reducing agent, and reacting the reduced cysteine ​​residue with the electrophile.

19. The method according to claim 18, wherein the azaphosphine reducing agent is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) or a derivative thereof.

20. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

21. Use of a pharmaceutical composition according to claim 20 in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and / or eczema.

22. Use of the pharmaceutical composition according to claim 20 in the preparation of a medicament for reducing at least one of food intake or body weight in a subject in need thereof.

23. Use of the pharmaceutical composition according to claim 20 in the preparation of a medicament for modulating Y2 receptor activity and / or GLP-1 receptor activity in a subject in need thereof.

24. Use according to any one of claims 21 to 23, wherein the pharmaceutical composition is administered via injection.

25. A kit comprising the conjugate according to any one of claims 1 to 16, preferably further comprising a device for injection.

26. A method for preparing a pharmaceutical composition comprising the conjugate according to any one of claims 1 to 16, the method comprising combining the conjugate with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

27. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 136 and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising the conjugate according to claim 27 and a pharmaceutically acceptable carrier.

29. Use of the pharmaceutical composition according to claim 28 in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, hypertension, cardiovascular risk factors associated with unmanaged cholesterol, cardiovascular risk factors associated with lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and / or eczema.

30. Use of the pharmaceutical composition of claim 28 in the preparation of a medicament for reducing at least one of food intake or body weight in a subject in need thereof.

31. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 113 and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

32. A pharmaceutical composition comprising the conjugate according to claim 31 and a pharmaceutically acceptable carrier.

33. Use of a pharmaceutical composition according to claim 32 in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, hypertension, cardiovascular risk factors associated with unmanaged cholesterol, cardiovascular risk factors associated with lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and / or eczema.

34. Use of the pharmaceutical composition of claim 32 in the preparation of a medicament for reducing at least one of food intake or body weight in a subject in need thereof.

35. A conjugate comprising a glucagon-like peptide 1 (GLP-1) fusion peptide coupled to a cyclic PYY peptide, wherein the GLP-1 fusion peptide comprises the amino acid sequence of SEQ ID NO: 131 and the cyclic PYY peptide comprises an amino acid sequence selected from SEQ ID NO: 24, 25, 27, 28, 29, 30, 33 or 34, or a pharmaceutically acceptable salt thereof.

36. A pharmaceutical composition comprising the conjugate according to claim 35 and a pharmaceutically acceptable carrier.

37. Use of a pharmaceutical composition according to claim 36 in the preparation of a medicament for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, hypertension, cardiovascular risk factors associated with unmanaged cholesterol, cardiovascular risk factors associated with lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), nephropathy and / or eczema.

38. Use of the pharmaceutical composition of claim 36 in the preparation of a medicament for reducing at least one of food intake or body weight in a subject in need thereof.

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